RNA interference mediating small RNA molecules

ABSTRACT

Double-stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a  Drosophila  in vitro system, we demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNP complex.

[0001] The present invention relates to sequence and structural featuresof double-stranded (ds)RNA molecules required to mediate target-specificnucleic acid modifications such as RNA-interference and/or DNAmethylation.

[0002] The term “RNA interference” (RNAi) was coined after the discoverythat injection of dsRNA into the nematode C. elegans leads to specificsilencing of genes highly homologous in sequence to the delivered dsRNA(Fire et al., 1998). RNAi was subsequently also observed in insects,frogs (Oelgeschlager et al., 2000), and other animals including mice(Svoboda et al., 2000; Wianny and Zernicka-Goetz, 2000) and is likely toalso exist in human. RNAi is closely linked to the post-transcriptionalgene-silencing (PTGS) mechanism of co-suppression in plants and quellingin fungi (Catalanotto et al., 2000; Cogoni and Macino, 1999; Dalmay etal., 2000, Ketting and Plasterk, 2000; Mourrain et al., 2000; Smardon etal., 2000) and some components of the RNAi machinery are also necessaryfor post-transcriptional silencing by co-suppression (Catalanotto etal., 2000; Dernburg et al., 2000; Ketting and Plasterk, 2000). The topichas also been reviewed recently (Bass, 2000; Bosher and Labouesse, 2000;Fire, 1999; Plasterk and. Ketting, 2000; Sharp, 1999; Sijen and Kooter,2000), see also the entire issue of Plant Molecular Biology, vol. 43,issue 2/3, (2000).

[0003] In plants, in addition to PTGS, introduced transgenes can alsolead to transcriptional gene silencing via RNA-directed DNA methylationof cytosines (see references in Wassenegger, 2000). Genomic targets asshort as 30 bp are methylated in plants in an RNA-directed manner(Pelissier, 2000). DNA methylation is also present in mammals.

[0004] The natural function of RNAi and co-suppression appears to beprotection of the genome against invasion by mobile genetic elementssuch as retrotransposons and viruses which produce aberrant RNA or dsRNAin the host cell when they become active (Jensen et al, 1999; Ketting etal., 1999; Ratcliff et al., 1999; Tabara et al., 1999). Specific mRNAdegradation prevents transposon and virus replication although someviruses are able to overcome or prevent this process by expressingproteins that suppress PTGS (Lucy et al.; 2000; Voinnet et al., 2000).

[0005] DsRNA triggers the specific degradation of homologous RNAs onlywithin the region of identity with the dsRNA (Zamore et al., 2000). ThedsRNA is processed to 21-23 nt RNA fragments and the target RNA cleavagesites are regularly spaced 21-23 nt apart. It has therefore beensuggested that the 21-23 nt fragments are the guide RNAs for targetrecognition (Zamore et al., 2000). These short RNAs were also detectedin extracts prepared from. D. melanogaster Schneider 2 cells which weretransfected with dsRNA prior to cell lysis (Hammond et al. 2000),however, the fractions that displayed sequence-specific nucleaseactivity also contained a large fraction of residual dsRNA. The role ofthe 21-23 nt fragments in guiding mRNA cleavage is further supported bythe observation that 21-23 nt fragments isolated from processed dsRNAare able, to some extent, to mediate specific mRNA degradation (Zamoreet al., 2000). RNA molecules of similar size also accumulate in planttissue that exhibits PTGS (Hamilton and Baulcombe, 1999).

[0006] Here, we use the established Drosophila in vitro system (Tuschlet al., 1999; Zamore et al., 2000) to further explore the mechanism ofRNAi. We demonstrate that short 21 and 22 nt RNAs, when base-paired with3′ overhanging ends, act as the guide RNAs for sequence-specific mRNAdegradation. Short 30 bp dsRNAs are unable to mediate RNAi in thissystem because they are no longer processed to 21 and 22 nt RNAs.Furthermore, we defined the target RNA cleavage sites relative to the 21and 22 nt short interfering RNAs (siRNAs) and provide evidence that thedirection of dsRNA processing determines whether a sense or an antisensetarget RNA can be cleaved by the produced siRNP endonuclease complex.Further, the siRNAs may also be important tools for transcriptionalmodulating, e.g. silencing of mammalian genes by guiding DNAmethylation.

[0007] Further experiments in human in vivo cell culture systems (HeLacells) show that double-stranded RNA molecules having a length ofpreferably from 19-25 nucleotides have RNAi activity. Thus, in contrastto the results from Drosophila also 24 and 25 nt long double-strandedRNA molecules are efficient for RNAi.

[0008] The object underlying the present invention is to provide novelagents capable of mediating target-specific RNA interference or othertarget-specific nucleic acid modifications such as DNA methylation, saidagents having an improved efficacy and safety compared to prior artagents.

[0009] The solution of this problem is provided by an isolateddouble-stranded RNA molecule, wherein each RNA strand has a length from19-25, particularly from 19-23 nucleotides, wherein said RNA molecule iscapable of mediating target-specific nucleic acid modifications,particularly RNA interference and/or DNA methylation. Preferably atleast one strand has a 3′-overhang from 1-5 nucleotides, more preferablyfrom 1-3 nucleotides and most preferably 2 nucleotides. The other strandmay be blunt-ended or has up to 6 nucleotides 3′ overhang. Also, if bothstrands of the dsRNA are exactly 21 or 22 nt, it is possible to observesome RNA interference when both ends are blunt (O nt overhang). The RNAmolecule is preferably a synthetic RNA molecule which is substantiallyfree from contaminants occurring in cell extracts, e.g. from Drosophilaembryos. Further, the RNA molecule is preferably substantially free fromany non-target-specific contaminants, particularly non-target-specificRNA molecules e.g. from contaminants occuring in cell extracts.

[0010] Further, the invention relates to the use of isolateddouble-stranded RNA molecules, wherein each RNA strand has a length from19-25 nucleotides, for mediating, target-specific nucleic acidmodifications, particularly RNAi, in mammalian cells, particularly inhuman cells.

[0011] Surprisingly, it was found that synthetic short double-strandedRNA molecules particularly with overhanging 3′-ends aresequence-specific mediators of RNAi and mediate efficient target-RNAcleavage, wherein the cleavage site is located near the center of theregion spanned by the guiding short RNA.

[0012] Preferably, each strand of the RNA molecule has a length from20-22 nucleotides (or 20-25 nucleotides in mammalian cells), wherein thelength of each strand May be the same or different. Preferably, thelength of the 3′-overhang reaches from 1-3 nucleotides, wherein thelength of the overhang may be the same or different for each strand. TheRNA-strands preferably have 3′-hydroxyl groups. The 5′-terminuspreferably comprises a phosphate, diphosphate, triphosphate or hydroxylgroup. The most effective dsRNAs are composed of two 21 nt strands whichare paired such that 1-3, particularly 2 nt 3′ overhangs are present onboth ends of the dsRNA.

[0013] The target RNA cleavage reaction guided by siRNAs is highlysequence-specific. However, not all positions of a siRNA contributeequally to target recognition. Mismatches in the center of the siRNAduplex are most critical and essentially abolish target RNA cleavage. Incontrast, the 3′ nucleotide of the siRNA strand (e.g. position 21) thatis complementary to the single-stranded target RNA, does not contributeto specificity of the target recognition. Further, the sequence of theunpaired 2-nt 3′ overhang of the siRNA strand with the same polarity asthe target RNA is not critical for target RNA cleavage as only theantisense siRNA strand guides target recognition. Thus, from thesingle-stranded overhanging nucleotides only the penultimate position ofthe antisense siRNA (e.g. position 20) needs to match the targeted sensemRNA.

[0014] Surprisingly, the double-stranded RNA molecules of the presentinvention exhibit a high in vivo stability in serum or in growth mediumfor cell cultures. In order to further enhance the stability, the3′-overhangs may be stablized against degradation, e.g. they may beselected such that they consist of purine nucleotides, particularlyadenosine or guanosine nucleotides. Alternatively, substitution ofpyrimidine nucleotides by modified analogues, e.g. substitution ofuridine 2 nt 3′ overhangs by 2′-deoxythymidine is tolerated and does notaffect the efficiency of RNA interference. The absence of a 2‘hydroxyl’significantly enhances the nuclease resistance of the overhang in tissueculture medium.

[0015] In an especially preferred embodiment of the present inventionthe RNA molecule may contain at least one modified nucleotide analogue.The nucleotide analogues may be located at positions where thetarget-specific activity, e.g. the RNAi mediating activity is notsubstantially effected, e.g. in a region at the 5′-end and/or the 3′-endof the double-stranded RNA molecule. Particularly, the overhangs may bestabilized by incorporating modified nucleotide analogues.

[0016] Preferred nucleotide analogues are selected from sugar- orbackbone-modified ribonucleotides. It should be noted, however, thatalso nucleobase-modified ribonucleotides, i.e. ribonucleotides,containing a non-naturally occurring nucleobase instead of a naturallyoccurring nucleobase such as uridines or cytidines modified at the5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosinesand guanosines modified at the 8-position, e.g. 8-bromo guanosine; deazanucleotides, e.g. 7-deaza-adenosine; O- and N-alkylated nucleotides,e.g. N6-methyl adenosine are suitable. In preferred sugar-modifiedribonucleotides the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or CN, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I. In preferredbackbone-modified ribonucleotides the phosphoester group connecting toadjacent ribonucleotides is replaced by a modified group, e.g. ofphosphothioate group. It should be noted that the above modificationsmay be combined.

[0017] The sequence of the double-stranded RNA molecule of the presentinvention has to have a sufficient identity to a nucleic acid targetmolecule in order to mediate target-specific RNAi and/or DNAmethylation. Preferably, the sequence has an identity of at least 50%,particularly of at least 70% to the desired target molecule in thedouble-stranded portion of the RNA molecule. More preferably, theidentity is at least 85% and most preferably 100% in the double-strandedportion of the RNA molecule. The identity of a double-stranded RNAmolecule to a predetermined nucleic acid target molecule, e.g. an mRNAtarget molecule may be determined as follows:$I = {\frac{n}{L} \times 100}$

[0018] wherein I is the identity in percent, n is the number ofidentical nucleotides in the double-stranded portion of the ds RNA andthe target and L is the length of the sequence overlap of thedouble-stranded portion of the dsRNA and the target.

[0019] Alternatively, the identity of the double-stranded RNA moleculeto the target sequence may also be defined including the 3′ overhang,particularly an overhang having a length from 1-3 nucleotides. In thiscase the sequence identity is preferably at least 50%, more preferablyat least 70% and most preferably at least 85% to the target sequence.For example, the nucleotides from the 3′ overhang and up to 2nucleotides from the 5′ and/or 3′ terminus of the double strand may bemodified without significant loss of activity.

[0020] The double-stranded RNA molecule of the invention may be preparedby a method comprising the steps:

[0021] (a) synthesizing two RNA strands each having a length from 19-25,e.g. from 19-23 nucleotides, wherein said RNA strands' are capable offorming a double-stranded RNA molecule, wherein preferably at least onestrand has a 3-overhang from 1-5 nucleotides,

[0022] (b) combining the synthesized RNA strands under conditions,wherein a double-stranded RNA molecule is formed, which is capable ofmediating target-specific nucleic acid modifications, particularly RNAinterference and/or DNA methylation.

[0023] Methods of synthesizing RNA molecules are known in the art. Inthis context, it is particularly referred to chemical synthesis methodsas described in Verma and Eckstein (1998).

[0024] The single-stranded RNAs can also be prepared by enzymatictranscription from synthetic DNA templates or from DNA plasmids isolatedfrom recombinant bacteria. Typically, phage RNA polymerases are usedsuch as T7, T3 or SP6 RNA polymerase (Milligan and Uhlenbeck (1989)).

[0025] A further aspect of the present invention relates to a method ofmediating target-specific nucleic acid modifications, particularly RNAinterference and/or DNA methylation in a cell or an organism comprisingthe steps:

[0026] (a) contacting the cell or organism with the double-stranded RNAmolecule of the invention under conditions wherein target-specificnucleic acid modifications may occur and

[0027] (b) mediating a target-specific nucleic acid modificiationeffected by the double-stranded RNA towards a target nucleic acid havinga sequence portion substantially corresponding to the double-strandedRNA.

[0028] Preferably the contacting step (a) comprises introducing thedouble-stranded RNA molecule into a target cell, e.g. an isolated targetcell, e.g. in cell culture, a unicellular microorganism or a target cellor a plurality of target cells within a multicellular organism. Morepreferably, the introducing step comprises a carrier-mediated delivery,e.g. by liposomal carriers or by injection.

[0029] The method of the invention may be used for determining thefunction of a gene in a cell or an organism or even for modulating thefunction of a gene in a cell or an organism, being capable of mediatingRNA interference. The cell is preferably a eukaryotic cell or a cellline, e.g. a plant cell or an animal cell, such as a mammalian cell,e.g. an embryonic cell, a pluripotent stem cell, a tumor cell, e.g. ateratocarcinoma cell or a virus-infected cell. The organism ispreferably a eukaryotic organism, e.g. a plant or an animal, such as amammal, particularly a human.

[0030] The target gene to which the RNA molecule of the invention isdirected may be associated with a pathological condition. For example,the gene may be a pathogen-associated gene, e.g. a viral gene, atumor-associated gene or an autoimmune disease-associated gene. Thetarget gene may also be a heterologous gene expressed in a recombinantcell or a genetically altered organism. By determinating or modulating,particularly, inhibiting the function of such a gene valuableinformation and therapeutic benefits in the agricultural field or in themedicine or veterinary medicine field may be obtained.

[0031] The dsRNA is usually administered as a pharmaceuticalcomposition. The administration may be carried out by known methods,wherein a nucleic acid is introduced into a desired target cell in vitroor in vivo. Commonly used gene transfer techniques include calciumphosphate, DEAE-dextran, electroporation and microinjection and viralmethods (Graham, F. L. and van der Eb, A. J. (1973) Virol. 52, 456;McCutchan, J. H. and Pagano, J. S. (1968), J. Natl Cancer Inst. 41, 351;Chu, G. et al (1987), Nucl. Acids Res. 15, 1311; Fraley, R. et al.(1980), J. Biol. Chem. 255, 10431; Capecchi, M. R. (1980), Cell 22,479). A recent addition to this arsenal of techniques for theintroduction of DNA into cells is the use of cationic liposomes(Feigner, P. L. et al. (1987), Proc. Natl. Acad. Sci USA 84, 7413).Commercially available cationic lipid formulations are e.g. Tfx 50(Promega) or Lipofectamin2000 (Life Technologies).

[0032] Thus, the invention also relates to a pharmaceutical compositioncontaining as an active agent at least one double-stranded RNA moleculeas described above and a pharmaceutical carrier. The composition may beused for diagnostic and for therapeutic applications in human medicineor in veterinary medicine.

[0033] For diagnostic or therapeutic applications, the composition maybe in form of a solution, e.g. an injectable solution, a cream,ointment, tablet, suspension or the like. The composition may beadministered in any suitable way, e.g. by injection, by oral, topical,nasal, rectal application etc. The carrier may be any suitablepharmaceutical carrier. Preferably, a carrier is used, which is capableof increasing the efficacy of the RNA molecules to enter thetarget-cells. Suitable examples of such carriers are liposomes,particularly cationic liposomes. A further preferred administrationmethod is injection.

[0034] A further preferred application of the RNAi method is afunctional analysis of eukaryotic cells, or eukaryotic non-humanorganisms, preferably mammalian cells or organisms and most preferablyhuman cells, e.g. cell lines such as HeLa or 293 or rodents, e.g. ratsand mice. By transfection with suitable double-stranded RNA moleculeswhich are homologous to a predetermined target gene or DNA moleculesencoding a suitable double-stranded RNA molecule a specific knockoutphenotype can be obtained in a target cell, e.g. in cell culture or in atarget organism. Surprisingly it was found that the presence of shortdouble-stranded RNA molecules does not result in an interferon responsefrom the host cell or host organism.

[0035] Thus, a further subject matter of the invention is a eukaryoticcell or a eukaryotic non-human organism exhibiting a targetgene-specific knockout phenotype comprising an at least partiallydeficient expression of at least one endogeneous target gene whereinsaid cell or organism is transfected with at least one double-strandedRNA molecule capable of inhibiting the expression of at least oneendogeneous target gene or with a DNA encoding at least one doublestranded RNA molecule capable of inhibiting the expression of at leastone endogeneous target gene. It should be noted that the presentinvention allows a target-specific knockout of several differentendogeneous genes due to the specificity of RNAi.

[0036] Gene-specific knockout phenotypes of cells or non-humanorganisms, particularly of human cells or non-human mammals may be usedin analytic procedures, e.g. in the functional and/or phenotypicalanalysis of complex physiological processes such as analysis of geneexpression profiles and/or proteomes. For example, one may prepare theknock-out phenotypes of human genes in cultured cells which are assumedto be regulators of alternative splicing processes. Among these genesare particularly the members of the SR splicing factor family, e.g.ASF/SF2, SC35, SRp20, SRp40 or SRp55. Further, the effect of SR proteinson the mRNA profiles of predetermined alternatively spliced genes suchas CD44 may be analysed. Preferably the analysis is carried out byhigh-throughput methods using oligonucleotide based chips.

[0037] Using RNAi based knockout technologies, the expression of anendogeneous target gene may be inhibited in a target cell or a targetorganism. The endogeneous gene may be complemented by an exogeneoustarget nucleic acid coding for the target protein or a variant ormutated form of the target protein, e.g. a gene or a cDNA, which mayoptionally be fused to a further nucleic acid sequence encoding adetectable peptide or polypeptide, e.g. an affinity tag, particularly amultiple affinity tag. Variants or mutated forms of the target genediffer from the endogeneous target gene in that they encode a geneproduct which differs from the endogeneous gene product on the aminoacid level by substitutions, insertions and/or deletions of single ormultiple amino acids. The variants or mutated forms may have the samebiological activity as the endogeneous target gene. On the other hand,the variant or mutated target gene may also have a biological activity,which differs from the biological activity of the endogeneous targetgene, e.g. a partially deleted activity, a completely deleted activity,an enhanced activity etc.

[0038] The complementation may be accomplished by coexpressing thepolypeptide encoded by the exogeneous nucleic acid, e.g. a fusionprotein comprising the target protein and the affinity tag and thedouble stranded RNA molecule for knocking out the endogeneous gene inthe target cell. This coexpression may be accomplished by using asuitable expression vector expressing both the polypeptide encoded bythe exogeneous nucleic acid, e.g. the tag-modified target protein andthe double stranded RNA molecule or alternatively by using a combinationof expression vectors. Proteins and protein complexes which aresynthesized de novo in the target cell will contain the exogeneous geneproduct, e.g. the modified fusion protein. In order to avoid suppressionof the exogeneous gene product expression by the RNAi duplex molecule,the nucleotide sequence encoding the exogeneous nucleic acid may bealtered on the DNA level (with or without causing mutations on the aminoacid level) in the part of the sequence which is homologous to thedouble stranded RNA molecule. Alternatively, the endogeneous target genemay be complemented by corresponding nucleotide sequences from otherspecies, e.g. from mouse.

[0039] Preferred applications for the cell or organism of the inventionis the analysis of gene expression profiles and/or proteomes. In anespecially preferred embodiment an analysis of a variant or mutant formof one or several target proteins is carried out, wherein said variantor mutant forms are reintroduced into the cell or organism by anexogeneous target nucleic acid as described above. The combination ofknockout of an endogeneous gene and rescue by using mutated, e.g.partially deleted exogeneous target has advantages compared to the useof a knockout cell. Further, this method is particularly suitable foridentifying functional domains of the target protein. In a furtherpreferred embodiment a comparison, e.g. of gene expression profilesand/or proteomes and/or phenotypic characteristics of at least two cellsor organisms is carried out. These organisms are selected from:

[0040] (i) a control cell or control organism without target geneinhibition,

[0041] (ii) a cell or organism with target gene inhibition and

[0042] (iii) a cell or organism with target gene inhibition plus targetgene complementation by an exogeneous target nucleic acid.

[0043] The method and cell of the invention are also suitable in aprocedure for identifying and/or characterizing pharmacological agents,e.g. identifying new pharmacological agents from a collection of testsubstances and/or characterizing mechanisms of action and/or sideeffects of known pharmacological agents.

[0044] Thus, the present invention also relates to a system foridentifying and/or characterizing pharmacological agents acting on atleast one target protein comprising:

[0045] (a) a eukaryotic cell or a eukaryotic non-human organism capableof expressing at least one endogeneous target gene coding for saidtarget protein,

[0046] (b) at least one double-stranded RNA molecule capable ofinhibiting the expression of said at least one endogeneous target gene,and

[0047] (c) a test substance or a collection of test substances whereinpharmacological properties of said test substance or said collection areto be identified and/or characterized.

[0048] Further, the system as described above preferably comprises:

[0049] (d) at least one exogeneous target nucleic acid coding for thetarget protein or a variant or mutated form of the target proteinwherein said exogeneous target nucleic acid differs from the endogeneoustarget gene on the nucleic acid level such thatthe expression of theexogeneous target nucleic acid is substantially less inhibited by thedouble stranded RNA molecule than the expression of the endogeneoustarget gene.

[0050] Furthermore, the RNA knockout complementation method may be usedfor preparative purposes, e.g. for the affinity purification of proteinsor protein complexes from eukaryotic cells, particularly mammalian cellsand more particularly human cells. In this embodiment of the invention,the exogeneous target nucleic acid preferably codes for a target proteinwhich is fused to an affinity tag.

[0051] The preparative method may be employed for the purification ofhigh molecular weight protein complexes which preferably have a mass of≧150 kD and more preferably of ≧500 kD and which optionally may containnucleic acids such as RNA. Specific examples are the heterotrimericprotein complex consisting of the 20 kD, 60 kD and 90 kD proteins of theU4/U6 snRNP particle, the splicing factor SF3b from the 17S U2 snRNPconsisting of 5 proteins having molecular weights of 14, 49, 120, 145and 155 kD and the 25S U4/U6/U5 tri-snRNP particle containing the U4, U5and U6 snRNA molecules and about 30 proteins, which has a molecularweight of about 1.7 MD.

[0052] This method is suitable for functional proteome analysis inmammalian cells, particularly human cells.

[0053] Further, the present invention is explained in more detail in thefollowing figures and examples.

FIGURE LEGENDS

[0054]FIG. 1: Double-stranded RNA as short as 38 bp can mediate RNAi.(A) Graphic representation of dsRNAs used for targeting Pp-luc mRNA.Three series of blunt-ended dsRNAs covering a range of 29 to 504 bp wereprepared. The position of the first nucleotide of the sense strand ofthe dsRNA is indicated relative to the start codon of Pp-luc mRNA (p1).(B) RNA interference assay (Tuschl et al., 1999). Ratios of targetPp-luc to control Rr-luc activity were normalized to a buffer control(black bar). DsRNAs (5 nM) were preincubated in Drosophila lysate for 15min at 25° C. prior to the addition of 7-methyl-guanosine-capped Pp-lucand Rr-luc mRNAs (˜50 pM). The incubation was continued for another hourand then analyzed by the dual luciferase assay (Promega). The data arethe average from at least four independent experiments±standarddeviation.

[0055]FIG. 2: A 29 bp dsRNA is no longer processed to 21-23 ntfragments. Time course of 21-23 mer formation from processing ofinternally ³²P labeled dsRNAs (5 nM) in the Drosophila lysate. Thelength and source of the dsRNA are indicated. An RNA size marker (M) hasbeen loaded in the left lane and the fragment sizes are indicated.Double bands at time zero are due to incompletely denatured dsRNA.

[0056]FIG. 3: Short dsRNAs cleave the mRNA target only once.

[0057] (A) Denaturing gel electrophoreses of the stable 5′ cleavageproducts produced by 1 h incubation of 10 nM sense or antisense RNA³²P-labeled at the cap with 10 nM dsRNAs of the p133 series inDrosophila lysate. Length markers were generated by partial nuclease T1digestion and partial alkaline hydrolysis (OH) of the cap-labeled targetRNA. The regions targeted by the dsRNAs are indicated as black bars onboth sides. The 20-23 nt spacing between the predominant cleavage sitesfor the 111 bp long dsRNA is shown. The horizontal arrow indicatesunspecific cleavage not due to RNAi. (B) Position of the cleavage siteson sense and antisense target RNAs. The sequences of the capped 177 ntsense and 180 nt antisense target RNAs are represented in antiparallelorientation such that complementary sequence are opposing each other.The region targeted by the different dsRNAs are indicated by differentlycolored bars positioned between sense and antisense target sequences.Cleavage sites are indicated by circles: large circle for strongcleavage, small circle for weak cleavage. The ³²P-radiolabeled phosphategroup is marked by an asterisk.

[0058]FIG. 4: 21 and 22 nt RNA fragments are generated by an RNaseIII-like mechanism.

[0059] (A) Sequences of ˜21 nt RNAs after dsRNA processing. The ˜21 ntRNA fragments generated by dsRNA processing were directionally clonedand sequenced. Oligoribonucleotides originating from the sense strand ofthe dsRNA are indicated as blue lines, those originating from theantisense strand as red lines. Thick bars are used if the same sequencewas present in multiple clones, the number at the right indicating thefrequency. The target RNA cleavage sites mediated by the dsRNA areindicated as orange circles, large circle for strong cleavage, smallcircle for weak cleavage (see FIG. 3B). Circles on top of the sensestrand indicated cleavage sites within the sense target and circles atthe bottom of the dsRNA indicate cleavage site in the antisense target.Up to five additional nucleotides were identified in ˜21 nt fragmentsderived from the 3′ ends of the dsRNA. These nucleotides are randomcombinations of predominantly C, G, or A residues and were most likelyadded in an untemplated fashion during T7 transcription of thedsRNA-constituting strands. (B) Two-dimensional TLC analysis of thenucleotide composition of ˜21 nt RNAs. The ˜21 nt RNAs were generated byincubation of internally radiolabeled 504 bp Pp-luc dsRNA in Drosophilalysate, gel-purified, and then digested to mononucleotides with nucleaseP1 (top row) or ribonuclease T2 (bottom row). The dsRNA was internallyradiolabeled by transcription in the presence of one of the indicatedα-³²P nucleoside triphosphates. Radioactivity was detected byphosphorimaging. Nucleoside 5′-monophosphates, nucleoside3′-monophosphates, nucleoside 5′,3′-diphosphates, and inorganicphosphate are indicated as pN, Np, pNp, and p_(i), respectively. Blackcircles indicate UV-absorbing spots from non-radioactive carriernucleotides. The 3′,5′-bis-phosphates (red circles) were identified byco-migration with radiolabeled standards prepared by 5′-phosphorylationof nucleoside 3′-mono-phosphates with T4 polynucleotide kinase andγ-³²P-ATP.

[0060]FIG. 5: Synthetic 21 and 22 nt RNAs Mediate Target RNA Cleavage.

[0061] (A) Graphic representation of control 52 bp dsRNA and synthetic21 and 22 nt dsRNAs. The sense strand of 21 and 22 nt short interferingRNAs (siRNAs) is shown blue, the antisense strand in red. The sequencesof the siRNAs were derived from the cloned fragments of 52 and 111 bpdsRNAs (FIG. 4A), except for the 22 nt antisense strand of duplex 5. ThesiRNAs in duplex 6 and 7 were unique to the 111 bp dsRNA processingreaction. The two 3′ overhanging nucleotides indicated in green arepresent in the sequence of the synthetic antisense strand of duplexes 1and 3. Both strands of the control 52 bp dsRNA were prepared by in vitrotranscription and a fraction of transcripts may contain untemplated 3′nucleotide addition. The target RNA cleavage sites directed by the siRNAduplexes are indicated as orange circles (see legend to FIG. 4A) andwere determined as shown in FIG. 5B. (B) Position of the cleavage siteson sense and antisense target RNAs. The target RNA sequences are asdescribed in FIG. 3B. Control 52 bp dsRNA (10 nM) or 21 and 22 nt RNAduplexes 1-7 (100 nM) were incubated with target RNA for 2.5 h at 25° C.in Drosophila lysate. The stable 5′ cleavage products were resolved onthe gel. The cleavage sites are indicated in FIG. 5A. The regiontargeted by the 52 bp dsRNA or the sense (s) or antisense (as) strandsare indicated by the black bars to the side of the gel. The cleavagesites are all located within the region of identity of the dsRNAs. Forprecise determination of the cleavage sites of the antisense strand, alower percentage gel was used.

[0062]FIG. 6: Long 3′ overhangs on short dsRNAs inhibit RNAi.

[0063] (A) Graphic representation of 52 bp dsRNA constructs. The 3′extensions of sense and antisense strand are indicated in blue and red,respectively. The observed cleavage sites on the target RNAs arerepresented as orange circles analogous to FIG. 4A and were determinedas shown in FIG. 6B. (B) Position of the cleavage sites on sense andantisense target RNAs. The target RNA sequences are as described in FIG.3B. DsRNA (10 nM) was incubated with target RNA for 2.5 h at 25° C. inDrosophila lysate. The stable 5′ cleavage products were resolved on thegel. The major cleavage sites are indicated with a horizontal arrow andalso represented in FIG. 6A. The region targeted by the 52 bp dsRNA isrepresented as black bar at both sides of the gel.

[0064]FIG. 7: Proposed Model for RNAi.

[0065] RNAi is predicted to begin with processing of dsRNA (sense strandin black, antisense strand in red) to predominantly 21 and 22 nt shortinterfering RNAs (siRNAs). Short overhanging 3′ nucleotides, if presenton the dsRNA, may be beneficial for processing of short dsRNAs. ThedsRNA-processing proteins, which remain to be characterized, arerepresented as green and blue ovals, and assembled on the dsRNA inasymmetric fashion. In our model, this is illustrated by binding of ahypothetical blue protein or protein domain with the siRNA strand in 3′to 5′ direction while the hypothetical green protein or protein domainis always bound to the opposing siRNA strand. These proteins or a subsetremain associated with the siRNA duplex and preserve its orientation asdetermined by the direction of the dsRNA processing reaction. Only thesiRNA sequence associated with the blue protein is able to guide targetRNA cleavage. The endonuclease complex is referred to as smallinterfering ribonucleoprotein complex or siRNP. It is presumed here,that the endonuclease that cleaves the dsRNA may also cleave the targetRNA, probably by temporarily displacing the passive siRNA strand notused for target recognition. The target RNA is then cleaved in thecenter of the region recognized by the sequence-complementary guidesiRNA.

[0066]FIG. 8: Reporter constructs and siRNA duplexes.

[0067] (a) The firefly (Pp-luc) and sea pansy (Rr-luc) luciferasereporter gene regions from plasmids pGL2-Control, pGL-3-Control andpRL-TK (Promega) are illustrated. SV40 regulatory elements, the HSVthymidine kinase promoter and two introns (lines) are indicated. Thesequence of GL3 luciferase is 95% identical to GL2, but RL is completelyunrelated to both. Luciferase expression from pGL2 is approx. 10-foldlower than from pGL3 in transfected mammalian cells. The region targetedby the siRNA duplexes is indicated as black bar below the coding regionof the luciferase genes. (b) The sense (top) and antisense (bottom)sequences of the siRNA duplexes targeting GL2, GL3 and RL luciferase areshown. The GL2 and GL3 siRNA duplexes differ by only 3 single nucleotidesubstitutions (boxed in gray). As unspecific control, a duplex with theinverted GL2 sequence, invGL2, was synthesized. The 2 nt 3′ overhang of2′-deoxythymidine is indicated as TT; uGL2 is similar to GL2 siRNA butcontains ribo-uridine 3′ overhangs.

[0068]FIG. 9: RNA interference by siRNA duplexes.

[0069] Ratios of target control luciferase were normalized to a buffercontrol (bu, black bars); gray bars indicate ratios of Photinus pyralis(Pp-luc) GL2 or GL3 luciferase to Renilla reniformis (Rr-luc) RLluciferase (left axis), white bars indicate RL to GL2 or GL3 ratios:(right axis). Panels a, c, e, g and i describe experiments performedwith the combination of pGL2-Control and pRL-TK reporter plasmids,panels b, d, f, h and j with pGL3-Control and pRL-TK reporter plasmids.The cell line used for the interference experiment is indicated at thetop of each plot. The ratios of Pp-luc/Rr-luc for the buffer control(bu) varied between 0.5 and 10 for pGL2/pRL and between 0.03 and 1 forpGL3/pRL, respectively, before normalization and between the variouscell lines tested. The plotted data were averaged from three independentexperiments±S.D.

[0070]FIG. 10: Effects of 21 nt siRNA, 50 bp and 500 bp dsRNAs onluciferase expression in HeLa cells.

[0071] The exact length of the long dsRNAs is indicated below the bars.Panels a, c and e describe experiments performed with pGL2-Control andpRL-TK reporter plasmids, panels b, d and f with pGL3-Control and pRL-TKreporter plasmids. The data were averaged from two in dependentexperiments±S.D. (a), (b) Absolute Pp-luc expression, plotted inarbitrary luminescence units. (c), (d) Rr-luc expression, plotted inarbitrary luminescence units. (e), (f) Ratios of normalized target tocontrol luciferase. The ratios of luciferase activity for siRNA duplexeswere normalized to a buffer control (bu, black bars); the luminescenceratios for 50 or 500 bp dsRNAs were normalized to the respective ratiosobserved for 50 and 500 bp dsRNA from humanized GFP (hG, black bars). Itshould be noted that the overall differences in sequences between the49- and 484 bp dsRNAs targeting GL2 and GL3 are not sufficient to conferspecificity between GL2 and GL3 targets (43 nt uninterrupted identity in49 bp segment, 239 nt longest uninterrupted identity in 484 bp segment).

[0072]FIG. 11: Variation of the 3′ overhang of duplexes of 21-nt siRNAs.

[0073] (A) Outline of the experimental strategy. The capped andpolyadenylated sense target mRNA is depicted and the relative positionsof sense and antisense siRNAs are shown. Eight series of duplexes,according to the eight different antisense strands were prepared. ThesiRNA sequences, and the number of overhanging nucleotides were changedin 1-nt steps. (B) Normalized relative luminescence of target luciferase(Photinus pyralis, Pp-luc) to control luciferase (Renilla reniformis,Rr-luc) in D. melanogaster embryo lysate in the presence of 5 nMblunt-ended dsRNAs. The luminescence ratios determined in the presenceof dsRNA were normalized to the ratio obtained for a buffer control (bu,black bar). Normalized ratios less than 1 indicate specificinterference. (C-J) Normalized interference ratios for eight series of21-nt siRNA duplexes. The sequences of siRNA duplexes are depicted abovethe bar graphs. Each panel shows the interference ratio for a set ofduplexes formed with a given antisense guide siRNA and 5 different sensesiRNAs. The number of overhanging nucleotides (3′ over-hang, positivenumbers; 0.5′ overhangs, negative numbers) is indicated on the x-axis.Data points were averaged from at least 3 independent experiments, errorbars represent standard deviations.

[0074]FIG. 12: Variation of the length of the sense strand of siRNAduplexes.

[0075] (A) Graphic representation of the experiment. Three 21-ntantisense strands were paired with eight sense siRNAs. The siRNAs werechanged in length at their 3′ end. The 3′ overhang of the antisensesiRNA was 1-nt (B), 2-nt (C), or 3-nt (D) while the sense siRNA overhangwas varied for each series. The sequences of the siRNA duplexes and thecorresponding interference ratios are indicated.

[0076]FIG. 13: Variation of the length of siRNA duplexes with preserved2-nt 3′ overhangs.

[0077] (A) Graphic representation of the experiment. The 21-nt siRNAduplex is identical in sequence to the one shown in FIG. 11H or 12C. ThesiRNA duplexes were extended to the 3′ side of the sense siRNA (B) orthe 5′ side of the sense siRNA (C). The siRNA duplex sequences and therespective interference ratios are indicated.

[0078]FIG. 14: Substitution of the 2′-hydroxyl groups of the siRNAribose residues.

[0079] The 2′-hydroxyl groups (OH) in the strands of siRNA duplexes werereplaced by 2′-deoxy (d) or 2′-O-methyl (Me). 2-nt and 4-nt 2′-deoxysubstitutions at the 3′-ends are indicated as 2-nt d and 4-nt d,respectively. Uridine residues were replaced by 2′-deoxy thymidine.

[0080]FIG. 15: Mapping of sense and antisense target RNA cleavage by21-nt siRNA duplexes with 2-nt 3′ overhangs.

[0081] (A) Graphic representation of ³²P (asterisk) cap-labelled senseand anti-sense target RNAs and siRNA duplexes. The position of sense andanti-sense target RNA cleavage is indicated by triangles on top andbelow the siRNA duplexes, respectively. (B) Mapping of target RNAcleavage sites. After 2 h incubation of 10 nM target with 100 nM siRNAduplex in D. melanogaster embryo lysate, the 5′ cap-labelled substrateand the 5′ cleavage products were resolved on sequencing gels. Lengthmarkers were generated by partial RNase T1 digestion (T1) and partialalkaline hydrolysis (OH—) of the target RNAs. The bold lines to the leftof the images indicate the region covered by the siRNA strands 1 and 5of the same orientation as the target.

[0082]FIG. 16: The 5′ end of a guide siRNA defines the position oftarget RNA cleavage.

[0083] (A, B) Graphic representation of the experimental strategy. Theantisense siRNA was the same in all siRNA duplexes, but the sense strandwas varied between 18 to 25 nt by changing the 3′ end (A) or 18 to 23 ntby changing the 5′ end (B). The position of sense and antisense targetRNA cleavage is indicated by triangles on top and below the siRNAduplexes, respectively. (C, D) Analysis of target RNA cleavage usingcap-labelled sense (top panel) or antisense (bottom panel) target RNAs.Only the caplabelled 5′ cleavage products are shown. The sequences ofthe siRNA duplexes are indicated, and the length of the sense siRNAstrands is marked on top of the panel. The control lane marked with adash in panel (C) shows target RNA incubated in absence of siRNAs.Markers were as described in FIG. 15. The arrows in (D), bottom panel,indicate the target RNA cleavage sites that differ by 1 nt.

[0084]FIG. 17: Sequence variation of the 3′ overhang of siRNA duplexes.

[0085] The 2-nt 3‘overhang’ (NN, in gray) was changed in sequence andcomposition as indicated (T, 2′-deoxythymidine, dG, 2′-deoxyguanosine;asterisk, wild-type siRNA duplex). Normalized interference ratios weredetermined as described in FIG. 11. The wild-type sequence is the sameas depicted in FIG. 14.

[0086]FIG. 18: Sequence specificity of target recognition.

[0087] The sequences of the mismatched siRNA duplexes are shown,modified sequence segments or single nucleotides are underlayed in gray.The reference duplex (ref) and the siRNA duplexes 1 to 7 contain2′-deoxythymidine 2-nt overhangs. The silencing efficiency of thethymidine-modified reference duplex was comparable to the wild-typesequence (FIG. 17). Normalized interference ratios were determined asdescribed in FIG. 11.

[0088]FIG. 19: Variation of the length of siRNA duplexes with preserved2-nt 3′ overhangs.

[0089] The siRNA duplexes were extended to the 3′ side of the sensesiRNA (A) or the 5′ side of the sense siRNA (B). The siRNA duplexsequences and the respective interference ratios are indicated. For HeLaSS6 cells, siRNA duplexes (0.84 μg) targeting GL2 luciferase weretransfected together with pGL2-Control and pRL-TK plasmids. Forcomparison, the in vitro RNAi activities of siRNA duplexes tested in D.melanogaster lysate are indicated.

EXAMPLE 1

[0090] RNA Interference Mediated by Small Synthetic RNAs

[0091] 1.1. Experimental Procedures

[0092] 1.1.1 In Vitro RNAi

[0093] In vitro RNAi and lysate preparations were performed as describedpreviously (Tuschl et al., 1999; Zamore et al., 2000). It is critical touse freshly dissolved creatine kinase (Roche) for optimal ATPregeneration. The RNAi translation assays (FIG. 1) were performed withdsRNA concentrations of 5 nM and an extended pre-incubation period of 15min at 25° C. prior to the addition of in vitro transcribed, capped andpolyadenylated Pp-luc and Rr-luc reporter mRNAs. The incubation wascontinued for 1 h and the relative amount of Pp-luc and Rr-luc proteinwas analyzed using the dual luciferase assay (Promega) and a Monolight3010C luminometer (PharMingen).

[0094] 1.1.2 RNA Synthesis

[0095] Standard procedures were used for in vitro transcription of RNAfrom PCR templates carrying T7 or SP6 promoter sequences, see forexample (Tuschl et al., 1998). Synthetic RNA was prepared using ExpediteRNA phosphoramidites (Proligo). The 3′ adapter oligonucleotide wassynthesized usingdimethoxytrityl-1,4-benzenedimethanol-succinyl-aminopropyl-CPG. Theoligoribonucleotides were deprotected in 3 ml of 32% ammonia/ethanol(3/1) for 4 h at 55° C. (Expedite RNA) or 16 h at 55° C. (3′ and 5′adapter DNA/RNA chimeric oligonucleotides) and then desilylated andgel-purified as described previously (Tuschl et al., 1993). RNAtranscripts for dsRNA preparation including long 3′ overhangs weregenerated from PCR templates that contained a T7 promoter in sense andan SP6 promoter in antisense direction. The transcription template forsense and antisense target RNA was PCR amplified withGCGTAATACGACTCACTATAGAACAATTGCTTTTACAG (underlined, T7 promoter) as 5′primer and ATTTAGGTGACACTATAGGCATAAAGAATTGAAGA (underlined, SP6promoter) as 3′ primer and the linearized Pp-luc plasmid (pGEM-lucsequence) (Tuschl et al., 1999) as template; the T7-transcribed senseRNA was 177 nt long with the Pp-luc sequence between pos. 113-273relative to the start codon and followed by 17 nt of the complement ofthe SP6 promoter sequence at the 3′ end. Transcripts for blunt-endeddsRNA formation were prepared by transcription from two different PCRproducts which only contained a single promoter sequence.

[0096] DsRNA annealing was carried out using a phenol/chloroformextraction. Equimolar concentration of sense and antisense RNA (50 nM to10 μM, depending on the length and amount available) in 0.3 M NaOAc (pH6) were incubated for 30 s at 90° C. and then extracted at roomtemperature with an equal volume of phenol/chloroform, and followed by achloroform extraction to remove residual phenol. The resulting dsRNA wasprecipitated by addition of 2.5-3 volumes of ethanol. The pellet wasdissolved in lysis buffer (100 mM KCl, 30 mM HEPES-KOH, pH 7.4, 2 mMMg(OAc)₂) and the quality of the dsRNA was verified by standard agarosegel electrophoreses in 1×TAE-buffer. The 52 bp dsRNAs with the 17 nt and20 nt 3′ overhangs (FIG. 6) were annealed by incubating for 1 min at 95°C., then rapidly cooled to 70° C. and followed by slow cooling to roomtemperature over a 3 h period (50 μl annealing reaction, 1 μM strandconcentration, 300 mM NaCl, 10 mM Tris-HCl, pH 7.5). The dsRNAs werethen phenol/chloroform extracted, ethanol-precipitated and dissolved inlysis buffer.

[0097] Transcription of internally ³²P-radiolabeled RNA used for dsRNApreparation (FIGS. 2 and 4) was performed using 1 mM ATP, CTP, GTP, 0.1or 0.2 mM UTP, and 0.2-0.3 μM-³²P-UTP (3000 Ci/mmol), or the respectiveratio for radiolabeled nucleoside triphosphates other than UTP. Labelingof the cap of the target RNAs was performed as described previously. Thetarget RNAs were gel-purified after cap-labeling.

[0098] 1.1.3 Cleavage Site Mapping

[0099] Standard RNAi reactions were performed by pre-incubating 10 nMdsRNA for 15 min followed by addition of 10 nM cap-labeled target RNA.The reaction was stopped after a further 2 h (FIG. 2A) or 2.5 hincubation (FIGS. 5B and 6B) by proteinase K treatment (Tuschl et al.,1999). The samples were then analyzed on 8 or 10% sequencing gels. The21 and 22 nt synthetic RNA duplexes were used at 100 nM finalconcentration (FIG. 5B).

[0100] 1.1.4 Cloning of ˜21 nt RNAs

[0101] The 21 nt RNAs were produced by incubation of radiolabeled dsRNAin Drosophila lysate in absence of target RNA (200 μl reaction, 1 hincubation, 50 nM dsP111, or 100 nM dsP52 or dsP39). The reactionmixture was subsequently treated with proteinase K (Tuschl et al., 1999)and the dsRNA-processing products were separated on a denaturing 15%polyacrylamide gel. A band, including a size range of at least 18 to 24nt, was excised, eluted into 0.3 M NaCl overnight at 4° C. and insiliconized tubes. The RNA was recovered by ethanol-precipitation anddephosphorylated (30 μl reaction, 30 min, 50° C., 10 U alkalinephosphatase, Roche). The reaction was stopped by phenol/chloroformextraction and the RNA was ethanol-precipitated. The 3′ adapteroligonucleotide (pUUUaaccgcatccttctcx: upper-case, RNA; lowercase, DNA;p, phosphate; x, 4-hydroxymethylbenzyl) was then ligated to thedephosphorylated ˜21 nt RNA (20 μl reaction, 30 min, 37° C., 5 μM 3′adapter, 50 mM Tris-HCl, pH 7.6, 10 mM MgCl, 0.2 mM ATP, 0.1 mg/mlacetylated BSA, 15% DMSO, 25 U T4 RNA ligase, Amersham-Pharmacia) (Panand Uhlenbeck, 1992). The ligation reaction was stopped by the additionof an equal volume of 8 M urea/50 mM EDTA stopmix and directly loaded ona 15% gel. Ligation yields were greater 50%. The ligation product wasrecovered from the gel and 5′-phosphorylated (20 μl reaction, 30 min,37° C., 2 mM ATP, 5 U T4 polynucleotide kinase, NEB). Thephosphorylation reaction was stopped by phenol/chloroform extraction andRNA was recovered by ethanol-precipitation. Next, the 5′ adapter(tactaatacgactcactAAA: uppercase, RNA; lowercase, DNA) was ligated tothe phosphorylated ligation product as described above. The new ligationproduct was gel-purified and eluted from the gel slice in the presenceof reverse transcription primer (GACTAGCTGGAATTCAAGGATGCGGTTAAA: bold,Eco RI site) used as carrier. Reverse transcription (15 μl reaction, 30min, 42° C., 150 U Superscript II reverse transcriptase, LifeTechnologies) was followed by PCR using as 5′ primerCAGCCAACGGAATTCATACGACTCACTAAA (bold, Eco RI site) and the 3′ RT primer.The PCR product was purified by phenol/chloroform extraction andethanol-precipitated. The PCR product was then digested with Eco RI(NEB) and concatamerized using T4 DNA ligase (high conc., NEB).Concatamers of a size range of 200 to 800 bp were separated on alow-melt agarose gel, recovered from the gel by a standard melting andphenol extraction procedure, and ethanol-precipitated. The unpaired endswere filled in by incubation with Taq polymerase under standardconditions for 15 min at 72° C. and the DNA product was directly ligatedinto the pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen).Colonies were screened using PCR and M13-20 and M13 Reverse sequencingprimers. PCR products were directly submitted for custom sequencing(Sequence Laboratories Göttingen GmbH, Germany). On average, four tofive 21mer sequences were obtained per clone.

[0102] 1.1.5 2D-TLC Analysis

[0103] Nuclease P1 digestion of radiolabeled, g61-purified siRNAs and2D-TLC was carried out as described (Zamore et al., 2000). Nuclease T2digestion was performed in 10 μl reactions for 3 h at 50° C. in 10 mMammonium acetate (pH 4.5) using 2 μg/μl carrier tRNA and 30 Uribonuclease T2 (Life Technologies). The migration of non-radioactivestandards was determined by UV shadowing. The identity ofnucleoside-3′,5′-disphosphates was confirmed by co-migration of the T2digestion products with standards prepared by 5′-³²P-phosphorylation ofcommercial nucleoside 3′-monophosphates using γ-³²P-ATP and T4polynucleotide kinase (data not shown).

[0104] 1.2 Results and Discussion

[0105] 1.2.1 Length Requirements for Processing of dsRNA to 21 and 22 ntRNA Fragments

[0106] Lysate prepared from D. melanogaster syncytial embryosrecapitulates RNAi in vitro providing a novel tool for biochemicalanalysis of the mechanism of RNAi (Tuschl et al., 1999; Zamore et al.,2000). In vitro and in vivo analysis of the length requirements of dsRNAfor RNAi has revealed that short dsRNA (<150 bp) are less effective thanlonger dsRNAs in degrading target mRNA (Caplen et al., 2000; Hammond etal., 2000; Ngo et al., 1998); Tuschl et al., 1999). The reasons forreduction in mRNA degrading efficiency are not understood. We thereforeexamined the precise length requirement of dsRNA for target RNAdegradation under optimized conditions in the Drosophila lysate (Zamoreet al., 2000). Several series of dsRNAs were synthesized and directedagainst firefly luciferase (Pp-luc) reporter RNA. The specificsuppression of target RNA expression was monitored by the dualluciferase assay (Tuschl et al., 1999) (FIGS. 1A and 1B). We detectedspecific inhibition of target RNA expression for dsRNAs as short as 38bp, but dsRNAs of 29 to 36 bp were not effective in this process. Theeffect was independent-of the target position and the degree ofinhibition of Pp-luc mRNA expression correlated with the length of thedsRNA, i.e. long dsRNAs were more effective than short dsRNAs.

[0107] It has been suggested that the 21-23 nt RNA fragments generatedby processing of dsRNAs are the mediators of RNA interference andco-suppression (Hamilton and Baulcombe, 1999; Hammond et al., 2000;Zamore et al., 2000). We therefore analyzed the rate of 21-23 ntfragment formation for a subset of dsRNAs ranging in size between 501 to29 bp. Formation of 21-23 nt fragments in Drosophila lysate (FIG. 2) wasreadily detectable for 39 to 501 bp long dsRNAs but was significantlydelayed for the 29 bp dsRNA. This observation is consistent with a roleof 21-23 nt fragments in guiding mRNA cleavage and provides anexplanation for the lack of RNAi by 30 bp dsRNAs. The length dependenceof 21-23 mer formation is likely to reflect a biologically relevantcontrol mechanism to prevent the undesired activation of RNAi by shortintramolecular base-paired structures of regular cellular RNAs.

[0108] 1.2.2 39 bp dsRNA Mediates Target RNA Cleavage at a Single Site

[0109] Addition of dsRNA and 5′-capped target RNA to the Drosophilalysate results in sequence-specific degradation of the target RNA(Tuschl et al., 1999). The target mRNA is only cleaved within the regionof identity with the dsRNA and many of the target cleavage sites wereseparated by 21-23 nt (Zamore et al., 2000). Thus, the number ofcleavage sites for a given dsRNA was expected to roughly correspond tothe length of the dsRNA divided by 21. We mapped the target cleavagesites on a sense and an antisense target RNA which was 5′ radiolabeledat the cap (Zamore et al., 2000) (FIGS. 3A and 3B). Stable 5′ cleavageproducts were separated on a sequencing gel and the position of cleavagewas determined by comparison with a partial RNase T1 and an alkalinehydrolysis ladder from the target RNA.

[0110] Consistent with the previous observation (Zamore et al., 2000),all target RNA cleavage sites were located within the region of identityto the dsRNA. The sense or the antisense traget was only cleaved once by39 bp dsRNA. Each cleavage site was located 10 nt from the 5′ end of theregion covered by the dsRNA (FIG. 3B). The 52 bp dsRNA, which shares thesame 5′ end with the 39 bp dsRNA, produces the same cleavage site on thesense target, located 10 nt from the 5′ end of the region of identitywith the dsRNA, in addition to two weaker cleavage sites 23 and 24 ntdownstream of the first site. The antisense target was only cleavedonce, again 10 nt from the 5′ end of the region covered by itsrespective dsRNA. Mapping of the cleavage sites for the 38 to 49 bpdsRNAs shown in FIG. 1 showed that the first and predominant cleavagesite was always located 7 to 10 nt downstream of the region covered bythe dsRNA (data not shown). This suggests that the point of target RNAcleavage is determined by the end of the dsRNA and could imply thatprocessing to 21-23 mers starts from the ends of the duplex.

[0111] Cleavage sites on sense and antisense target for the longer 111bp dsRNA were much more frequent than anticipated and most of themappear in clusters separated by 20 to 23 nt (FIGS. 3A and 3B). As forthe shorter dsRNAs, the first cleavage site on the sense target is 10 ntfrom the 5′ end of the region spanned by the dsRNA, and the firstcleavage site on the antisense target is located 9 nt from the 5′ end ofregion covered by the dsRNA. It is unclear what causes this disorderedcleavage, but one possibility could be that longer dsRNAs may not onlyget processed from the ends but also internally, or there are somespecificity determinants for dsRNA processing which we do not yetunderstand. Some irregularities to the 21-23 nt spacing were alsopreviously noted (Zamore et al., 2000). To better understand themolecular basis of dsRNA processing and target RNA recognition, wedecided to analyze the sequences of the 21-23 nt fragments generated byprocessing of 39, 52, and 111 bp dsRNAs in the Drosophila lysate.

[0112] 1.2.3dsRNA is Processed to 21 and 22 nt RNAs by an RNase III-LikeMechanism

[0113] In order to characterize the 21-23 nt RNA fragments we examinedthe 5′ and 3′ termini of the RNA fragments. Periodate oxidation ofgel-purified 21-23 nt RNAs followed by 13-elimination indicated thepresence of a terminal 2′ and 3′ hydroxyl groups. The 21-23 mers werealso responsive to alkaline phosphatase treatment indicating thepresence of a 5′ terminal phosphate group. The presence of 5′ phosphateand 3′ hydroxyl termini suggests that the dsRNA could be processed by anenzymatic activity similar to E. coli RNase III (for reviews, see (Dunn,1982; Nicholson, 1999; Robertson, 1990; Robertson, 1982)).

[0114] Directional cloning of 21-23 nt RNA fragments was performed byligation of a 3′ and 5′ adapter oligonucleotide to the purified 21′-23mers using T4 RNA ligase. The ligation products were reversetranscribed, PCR-amplified, concatamerized, cloned, and sequenced. Over220 short RNAs were sequenced from dsRNA processing reactions of the 39,52 and 111 bp dsRNAs (FIG. 4A). We found the following lengthdistribution: 1% 18 nt, 5% 19 nt, 12% 20 nt, 45% 21 nt, 28% 22 nt, 6% 23nt, and 2% 24 nt. Sequence analysis of the 5′ terminal nucleotide of theprocessed fragments indicated that oligonucleotides with a 5′ guanosinewere underrepresented. This bias was most likely introduced by T4 RNAligase which discriminates against 5′ phosphorylated guanosine as donoroligonucleotide; no significant sequence bias was seen at the 3′ end.Many of the ˜21 nt fragments derived from the 3′ ends of the sense orantisense strand of the duplexes include 3′ nucleotides that are derivedfrom untemplated addition of nucleotides during RNA synthesis using T7RNA polymerase. Interestingly, a significant number of endogenousDrosophila ˜21 nt RNAs were also cloned, some of them from LTR andnon-LTR retrotransposons (data not shown). This is consistent with apossible role for RNAi in transposon silencing.

[0115] The ˜21 nt RNAs appear in clustered groups (FIG. 4A) which coverthe entire dsRNA sequences. Apparently, the processing reaction cuts thedsRNA by leaving staggered 3′ ends, another characteristic of RNase IIIcleavage. For the 39 bp dsRNA, two clusters of ˜21 nt RNAs were foundfrom each dsRNA-constituting strand including overhanging 3′ ends, yetonly one cleavage site was detected on the sense and antisense target(FIGS. 3A and 3B). If the ˜21 nt fragments were present assingle-stranded guide RNAs in a complex that mediates mRNA degradation,it could be assumed that at least two target cleavage sites exist, butthis was not the case. This suggests that the ˜21 nt RNAs may be presentin double-stranded form in the endonuclease complex but that only one ofthe strands can be used for target RNA recognition and cleavage. The useof only one of the ˜21 nt strands for target cleavage may simply bedetermined by the orientation in which the ˜21 nt duplex is bound to thenuclease complex. This orientation is defined by the direction in whichthe original dsRNA was processed.

[0116] The ˜21mer clusters for the 52 bp and 111 bp dsRNA are less welldefined when compared to the 39 bp dsRNA. The clusters are spread overregions of 25 to 30 nt most likely representing several distinctsubpopulations of ˜21 nt duplexes and therefore guiding target cleavageat several nearby sites. These cleavage regions are still predominantlyseparated by 20 to 23 nt intervals. The rules determining how regulardsRNA can be processed to ˜21 nt fragments are not yet understood, butit was previously observed that the approx. 21-23 nt spacing of cleavagesites could be altered by a run of uridines (Zamore et al., 2000). Thespecificity of dsRNA cleavage by E. coli RNase III appears to be mainlycontrolled by antideterminants, i.e. excluding some specific base-pairsat given positions relative to the cleavage site (Zhang and Nicholson,1997).

[0117] To test whether sugar-, base- or cap-modification were present inprocessed ˜21 nt RNA fragments, we incubated radiolabeled 505 bp Pp-lucdsRNA in lysate for 1 h, isolated the −21 nt products, and digested itwith P1 or T2 nuclease to mononucleotides. The nucleotide mixture wasthen analyzed by 2D thin-layer chromatography (FIG. 4B). None of thefour natural ribonucleotides were modified as indicated by P1 or T2digestion. We have previously analyzed adenosine to inosine conversionin the ˜21 nt fragments (after a 2 h incubation) and detected a smallextent (˜0.7%) deamination (Zamore et al., 2000); shorter incubation inlysate (1 h) reduced this inosine fraction to barely detectable levels.RNase T2, which cleaves 3′ of the phosphodiester linkage, producednucleoside 3′-phosphate and nucleoside 3′,5′-diphosphate, therebyindicating the presence of a 5′-terminal monophosphate. All fournucleoside 3′,5′-diphosphates were detected and suggest that theinternucleotidic linkage was cleaved with little or nosequence-specificity. In summary, the ˜21 nt fragments are unmodifiedand were generated from dsRNA such that 5′-monophosphates and3′-hydroxyls were present at the 5′-end.

[0118] 1.2.4 Synthetic 21 and 22 nt RNAs Mediate Target RNA Cleavage

[0119] Analysis of the products of dsRNA processing indicated that the˜21 nt fragments are generated by a reaction with all thecharacteristics of an RNase III cleavage reaction (Dunn, 1982;Nicholson, 1999; Robertson, 1990; Robertson, 1982). RNase III makes twostaggered cuts in both strands of the dsRNA, leaving a 3′ overhang ofabout 2 nt. We chemically synthesized 21 and 22 nt RNAs, identical insequence to some of the cloned ˜21 nt fragments, and tested them fortheir ability to mediate target RNA degradation (FIGS. 5A and 5B). The21 and 22 nt RNA duplexes were incubated at 100 nM concentrations in thelysate, a 10-fold higher concentrations than the 52 bp control dsRNA.Under these conditions, target RNA cleavage is readily detectable.Reducing the concentration of 21 and 22 nt duplexes from 100 to 10 nMdoes still cause target RNA cleavage. Increasing the duplexconcentration from 100 nM to 1000 nM however does not further increasetarget cleavage, probably due to a limiting protein factor within thelysate.

[0120] In contrast to 29 or 30 bp dsRNAs that did not mediate RNAi, the21 and 22 nt dsRNAs with overhanging 3′ ends of 2 to 4 nt mediatedefficient degradation of target RNA (duplexes 1, 3, 4, 6, FIGS. 5A and5B). Bluntended 21 or 22 nt dsRNAs (duplexes 2, 5, and 7, FIGS. 5A and5B) were reduced in their ability to degrade the target and indicatethat overhanging 3′ ends are critical for reconstitution of theRNA-protein nuclease complex. The single-stranded overhangs may berequired for high affinity binding of the ˜21 nt duplex to the proteincomponents. A 5′ terminal phosphate, although present after dsRNAprocessing, was not required to mediate target RNA cleavage and wasabsent from the short synthetic RNAs.

[0121] The synthetic 21 and 22 nt duplexes guided cleavage of sense aswell as antisense targets within the region covered by the short duplex.This is an important result considering that a 39 bp dsRNA which formstwo pairs of clusters of ˜21 nt fragments (FIG. 2), cleaved sense orantisense target only once and not twice. We interpret this result bysuggesting that only one of two strands present in the ˜21 nt duplex isable to guide target RNA cleavage and that the orientation of the ˜21 ntduplex in the nuclease complex is determined by the initial direction ofdsRNA processing. The presentation of an already perfectly processed ˜21nt duplex to the in vitro system however does allow formation of theactive sequence-specific nuclease complex with two possible orientationsof the symmetric RNA duplex. This results in cleavage of sense as wellas antisense target within the region of identity with the 21 nt RNAduplex.

[0122] The target cleavage site is located 11 or 12 nt downstream of thefirst nucleotide that is complementary to the 21 or 22 nt guidesequence, i.e. the cleavage site is near center of the region covered bythe 21 or 22 nt RNAs (FIGS. 4A and 4B). Displacing the sense strand of a22 nt duplex by two nucleotides (compare duplexes 1 and 3 in FIG. 5A)displaced the cleavage site of only the antisense target by twonucleotides. Displacing both sense and antisense strand by twonucleotides shifted both cleavage sites by two nucleotides (compareduplexes 1 and 4). We predict that it will be possible to design a pairof 21 or 22 nt RNAs to cleave a target RNA at almost any given position.

[0123] The specificity of target RNA cleavage guided by 21 and 22 ntRNAs appears exquisite as no aberrant cleavage sites are detected (FIG.5B). It should however be noted, that the nucleotides present in the 3′overhang of the 21 and 22 nt RNA duplex may contribute less to substraterecognition than the nucleotides near the cleavage site. This is basedon the observation that the 3′ most nucleotide in the 3′ overhang of theactive duplexes 1 or 3 (FIG. 5A) is not complementary to the target. Adetailed analysis of the specificity of RNAi can now be readilyundertaken using synthetic 21′ and 22 nt RNAs.

[0124] Based on the evidence that synthetic 21 and 22 nt RNAs withoverhanging 3′ ends mediate. RNA interference, we propose to name the˜21 nt RNAs “short interfering RNAs” or siRNAs and the respectiveRNA-protein complex a “small interfering ribonucleoprotein particle” orsiRNP.

[0125] 1.2.5 3′ Overhangs of 20 nt on Short dsRNAs Inhibit RNAi.

[0126] We have shown that short blunt-ended dsRNAs appear to beprocessed from the ends of the dsRNA. During our study of the lengthdependence of dsRNA in RNAi, we have also analyzed dsRNAs with 17 to 20nt overhanging 3′ ends and found to our surprise that they were lesspotent than blunt-ended dsRNAs. The inhibitory effect of long 3′ endswas particularly pronounced for dsRNAs up to 100 bp but was lessdramatic for longer dsRNAs. The effect was not due to imperfect dsRNAformation based on native gel analysis (data not shown). We tested ifthe inhibitory effect of long overhanging 3′ ends could be used as atool to direct dsRNA processing to only one of the two ends of a shortRNA duplex.

[0127] We synthesized four combinations of the 52 bp model dsRNA,bluntended, 3′ extension on only the sense strand, 3′ extension on onlythe antisense strand, and double 3′ extension on both strands, andmapped the target RNA cleavage sites after incubation in lysate (FIGS.6A and 6B). The first and predominant cleavage site of the sense targetwas lost when the 3′ end of the antisense strand of the duplex wasextended, and vice versa, the strong cleavage site of the antisensetarget was lost when the 3′ end of sense strand of the duplex wasextended. 3′ Extensions on both strands rendered the 52 bp dsRNAvirtually inactive. One explanation for the dsRNA inactivation by ˜20 nt3′ extensions could be the association of single-stranded RNA-bindingproteins which could interfere with the association of one of thedsRNA-processing factors at this end. This result is also consistentwith our model where only one of the strands of the siRNA duplex in theassembled siRNP is able to guide target RNA cleavage. The orientation ofthe strand that guides RNA cleavage is defined by the direction of thedsRNA processing reaction. It is likely that the presence of 3′staggered ends may facilitate the assembly of the processing complex. Ablock at the 3′ end of the sense strand will only permit dsRNAprocessing from the opposing 3′ end of the antisense strand. This inturn generates siRNP complexes in which only the antisense strand of thesiRNA duplex is able to guide sense target RNA cleavage. The same istrue for the reciprocal situation.

[0128] The less pronounced inhibitory effect of long 3′ extensions inthe case of longer dsRNAs (≧500 bp, data not shown) suggests to us thatlong dsRNAs may also contain internal dsRNA-processing signals or mayget processed cooperatively due to the association of multiple cleavagefactors.

[0129] 1.2.6 A Model for dsRNA-Directed mRNA Cleavage

[0130] The new biochemical data update the model for how dsRNA targetsmRNA for destruction (FIG. 7). Double-stranded RNA is first processed toshort RNA duplexes of predominantly 21 and 22 nt in length and withstaggered 3′ ends similar to an RNase III-like reaction (Dunn, 1982;Nicholson, 1999; Robertson, 1982). Based on the 21-23 nt length of theprocessed RNA fragments it has already been speculated that an RNaseIII-like activity may be involved in RNAi (Bass, 2000). This hypothesisis further supported by the presence of 5′ phosphates and 3′ hydroxylsat the termini of the siRNAs as observed in RNase III reaction products(Dunn, 1982; Nicholson, 1999). Bacterial RNase III and the eukaryotichomologs Rnt1p in S. cerevisiae and Pac1p in S. pombe have been shown tofunction in processing of ribosomal RNA as well as snRNA and snoRNAs(see for example Chanfreau et al., 2000).

[0131] Little is known about the biochemistry of RNase III homologs fromplants, animals or human. Two families of RNase III enzymes have beenidentified predominantly by database-guided sequence analysis or cloningof cDNAs. The first RNase III family is represented by the 1327 aminoacid long D. melanogaster protein drosha (Acc. AF116572). The C-terminusis composed of two RNase III and one dsRNA-binding domain and theN-terminus is of unknown function. Close homologs are also found in C.elegans (Acc. AF160248) and human (Acc. AF189011.) (Filippov et al.,2000; Wu et al., 2000). The drosha-like human RNase III was recentlycloned and characterized (Wu et al., 2000). The gene is ubiquitouslyexpressed in human tissues and cell lines, and the protein is localizedin the nucleus and the nucleolus of the cell. Based-on results inferredfrom antisense inhibition studies, a role of this protein for rRNAprocessing was suggested. The second class is represented by the C.elegans gene K12H4.8 (Acc. S44849) coding for a 1822 amino acid longprotein. This protein has an N-terminal RNA helicase motif which isfollowed by 2 RNase III catalytic domains and a dsRNA-binding motif,similar to the drosha RNase III family. There are close homologs in S.pombe (Acc. Q09884), A. thaliana (Acc. AF187317), D. melanogaster (Acc.AE003740), and human (Acc. AB028449) (Filippov et al., 2000; Jacobsen etal., 1999; Matsuda et al., 2000). Possibly the K12H4.8 RNaseIII/helicase is the likely candidate to be involved in RNAi.

[0132] Genetic screens in C. elegans identified rde-1 and rde-4-asessential for activation of RNAi without an effect on transposonmobilization or co-suppression (Dernburg et al., 2000; Grishok et al.,2000; Ketting and Plasterk, 2000; Tabara et al., 1999). This led to thehypothesis that these genes are important for dsRNA processing but arenot involved in mRNA target degradation. The function of both genes isas yet unknown, the rde-1 gene product is a member of a family ofproteins similar to the rabbit protein elF2C (Tabara et al., 1999), andthe sequence of rde-4 has not yet been described. Future biochemicalcharacterization of these proteins should reveal their molecularfunction.

[0133] Processing to the siRNA duplexes appears to start from the endsof both blunt-ended dsRNAs or dsRNAs with short (1-5 nt) 3′ overhangs,and proceeds in approximately 21-23 nt steps. Long (˜20 nt) 3′ staggeredends on short dsRNAs suppress RNAi, possibly through interaction withsingle-stranded RNA-binding proteins. The suppression of RNAi bysingle-stranded regions flanking short dsRNA and the lack of siRNAformation from short 30 bp dsRNAs may explain why structured regionsfrequently encountered in mRNAs do not lead to activation of RNAi.

[0134] Without wishing to be bound by theory, we presume that thedsRNA-processing proteins or a subset of these remain associated withthe siRNA duplex after the processing reaction. The orientation of thesiRNA duplex relative to these proteins determines which of the twocomplementary strands functions in guiding, target RNA degradation.Chemically synthesized siRNA duplexes guide cleavage of sense as well asantisense target RNA as they are able to associate with the proteincomponents in either of the two possible orientation.

[0135] The remarkable finding that synthetic 21 and 22 nt siRNA duplexescan be used for efficient mRNA degradation provides new tools forsequence-specific regulation of gene expression in functional genomicsas well as biomedical studies. The siRNAs may be effective in mammaliansystems where long dsRNAs cannot be used due to the activation of thePKR response (Clemens, 1997). As such, the siRNA duplexes represent anew alternative to antisense or ribozyme therapeutics.

EXAMPLE 2

[0136] RNA Interference in Human Tissue Cultures

[0137] 2.1 Methods

[0138] 2.1.1 RNA preparation

[0139] 21 nt RNAs were chemically synthesized using Expedite RNAphosphoramidites and thymidine phosphoramidite (Proligo, Germany).Synthetic oligonucleotides were deprotected and gel-purified (Example1), followed by Sep-Pak C18 cartridge (Waters, Milford, Mass., USA)purification (Tuschl, 1993). The siRNA sequences targeting GL2 (Acc.X65324) and GL3 luciferase (Acc. U47296) corresponded to the codingregions 153-173 relative to the first nucleotide of the start codon,siRNAs targeting RL (Acc. AF025846) corresponded to region 119-129 afterthe start codon. Longer RNAs were transcribed with T7 RNA polymerasefrom PCR products, followed by gel and Sep-Pak purification. The 49 and484 bp GL2 or GL3 dsRNAs corresponded to position 113-161 and 113-596,respectively, relative to the start of translation; the 50 and 501 bp RLdsRNAs corresponded to position 118-167 and 118-618, respectively. PCRtemplates for dsRNA synthesis targeting humanized GFP (hG) wereamplified from pAD3 (Kehlenbach, 1998), whereby 50 and 501 bp hG dsRNAcorresponded to position 118-167 and 118-618, respectively, to the startcodon.

[0140] For annealing of siRNAs, 20 μM single strands were incubated inannealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2mM magnesium acetate) for 1 min at 90° C. followed by 1 h at 37° C. The37° C. incubation step was extended overnight for the 50 and 500 bpdsRNAs and these annealing reactions were performed at 8.4 μM and 0.84μM strand concentrations, respectively.

[0141] 2.1.2. Cell Culture

[0142] S2 cells were propagated in Schneider's Drosophila medium (LifeTechnologies) supplemented with 10% FBS, 100 units/ml penicillin and 100μg/ml streptomycin at 25° C. 293, NIH/3T3, HeLa S3, COS-7 cells weregrown at 37° C. in Dulbecco's modified Eagle's medium supplemented with10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. Cells wereregularly passaged to maintain exponential growth. 24 h beforetransfection at approx. 80% confluency, mammalian cells were trypsinizedand diluted 1:5 with fresh medium without antibiotics (1-3×10⁵ cells/ml)and transferred to 24-well plates (500 μl/well). S2 cells were nottrypsinized before split-ting. Transfection was carried out withLipofectamine 2000 reagent (Life Technologies) as described by themanufacturer for adherent cell lines. Per well, 1.0 μg pGL2-Control(Promega) or pGL3-Control (Promega), 0.1 μg pRL-TK (Promega) and 0.28 μgsiRNA duplex or dsRNA, formulated into liposomes, were applied; thefinal volume was 600 μl per well. Cells were incubated 20 h aftertransfection and appeared healthy thereafter. Luciferase expression wassubsequently monitored with the Dual luciferase assay (Promega).Transfection efficiencies were determined by fluorescence microscopy formammalian cell lines after co-transfection of 1.1 μg hGFP-encoding pAD3and 0.28 μg invGL2 in GL2 siRNA and were 70-90%. Reporter plasmids wereamplified in XL-1 Blue (Stratagene) and purified using the QiagenEndoFree Maxi Plasmid Kit.

[0143] 2.2 Results and Discussion

[0144] To test whether siRNAs are also capable of mediating RNAi intissue culture, we synthesized 21 nt siRNA duplexes with symmetric 2 nt3′ overhangs directed against reporter genes coding for sea pansy(Renilla reniformis) and two sequence variants of firefly (Photinuspyralis, GL2 and GL3) luciferases (FIG. 8a, b). The siRNA duplexes wereco-transfected with the reporter plasmid combinations pGL2/pRL orpGL3/pRL into D. melanogaster Schneider S2 cells or mammalian cellsusing cationic liposomes. Luciferase activities were determined 20 hafter transfection. In all cell lines tested, we observed specificreduction of the expression of the reporter genes in the presence ofcognate siRNA duplexes (FIG. 9a-j). Remarkably, the absolute luciferaseexpression levels were unaffected by non-cognate siRNAs, indicating theabsence of harmful side effects by 21 nt RNA duplexes (e.g. FIG. 10a-dfor HeLa cells). In D. melanogaster S2 cells (FIG. 9a, b), the specificinhibition of luciferases was complete. In mammalian cells, where thereporter genes were 50- to 100-fold stronger expressed, the specificsuppression was less complete (FIG. 9c-j). GL2 expression was reduced 3-to 12-fold, GL3 expression 9- to 25-fold and RL expression 1- to 3-fold,in response to the cognate siRNAs. For 293 cells, targeting of RLluciferase by RL siRNAs was ineffective, although GL2 and GL3 targetsresponded specifically (FIG. 9i, j). The lack of reduction of RLexpression in 293 cells may be due to its 5- to 20-fold higherexpression compared to any other mammalian cell line tested and/or tolimited accessibility of the target sequence due to RNA secondarystructure or associated proteins. Nevertheless, specific targeting ofGL2 and GL3 luciferase by the cognate siRNA duplexes indicated that RNAiis also functioning in 293 cells.

[0145] The 2 nt 3′ overhang in all siRNA duplexes, except for uGL2, wascomposed of (2′-deoxy) thymidine. Substituion of uridine by thymidine inthe 3′ overhang was well tolerated in the D. melanogaster in vitro sytemand the sequence of the overhang was uncritical for target recognition.The thymidine overhang was chosen, because it is supposed to enhancenuclease resistance of siRNAs in the tissue culture medium and withintransfected cells. Indeed, the thymidine-modified GL2 siRNA was slightlymore potent than the unmodified uGL2 siRNA in all cell lines tested(FIG. 9a, c, e, g, i). It is conceivable that further modifications ofthe 3′ overhanging nucleotides may provide additional benefits to thedelivery and stability of siRNA duplexes.

[0146] In co-transfection experiments, 25 nM siRNA duplexes with respectto the final volume of tissue culture medium were used (FIG. 9, 10).Increasing the siRNA concentration to 100 nM did not enhance thespecific silencing effects, but started to affect transfectionefficiencies due to competition for liposome encapsulation betweenplasmid DNA and siRNA (data not shown). Decreasing the siRNAconcentration to 1.5 nM did not reduce the specific silencing effect(data not shown), even though the siRNAs were now only 2- to 20-foldmore concentrated than the DNA plasmids. This indicates that siRNAs areextraordinarily powerful reagents for mediating gene silencing and thatsiRNAs are effective at concentrations that are several orders ofmagnitude below the concentrations applied in conventional antisense orribozyme gene targeting experiments.

[0147] In order to monitor the effect of longer dsRNAs on mammaliancells, 50 and 500 bp dsRNAs cognate to the reporter genes were prepared.As nonspecific control, dsRNAs from humanized GFP (hG) (Kehlenbach,1998) was used. When dsRNAs were co-transfected, in identical amounts(not concentrations) to the siRNA duplexes, the reporter gene expressionwas strongly and unspecifically reduced. This effect is illustrated forHeLa cells as a representative example (FIG. 10a-d). The absoluteluciferase activities were decreased unspecifically 10- to 20-fold by 50bp dsRNA and 20- to 200-fold by 500 bp dsRNA co-transfection,respectively. Similar unspecific effects were observed for COS-7 andNIH/3T3 cells. For 293 cells, a 10- to 20-fold unspecific reduction wasobserved only for 500 bp dsRNAs. Unspecific reduction in reporter geneexpression by dsRNA >30 bp was expected as part of the interferonresponse.

[0148] Surprisingly, despite the strong unspecific decrease in reportergene expression, we reproducibly detected additional sequence-specific,dsRNA-mediated silencing. The specific silencing effects, however, wereonly apparent when the relative reporter gene activities were normalizedto the hG dsRNA controls (FIG. 10e, f). A 2- to 10-fold specificreduction in response to cognate dsRNA was observed, also in the otherthree mammalian cell lines tested (data not shown). Specific silencingeffects with dsRNAs (356-1662 bp) were previously reported in CHO-K1cells, but the amounts of dsRNA required to detect a 2- to 4-foldspecific reduction were about 20-fold higher than in our experiments(Ui-Tei, 2000). Also CHO-K1 cells appear to be deficient in theinterferon response. In another report, 293, NIH/3T3 and BHK-21 cellswere tested for RNAi using luciferase/lacZ reporter combinations and 829bp specific lacZ or 717 bp unspecific GFP dsRNA (Caplen, 2000). Thefailure of detecting RNAi in this case may be due to the less sensitiveluciferase/lacZ reporter assay and the length differences of target andcontrol dsRNA. Taken together, our results indicate that RNAi is activein mammalian cells, but that the silencing effect is difficult todetect, if the interferon system is activated by dsRNA >30 bp.

[0149] In summary, we have demonstrated for the first timesiRNA-mediated gene silencing in mammalian cells. The use of shortsiRNAs holds great promise for inactivation of gene function in humantissue culture and the development of gene-specific therapeutics.

EXAMPLE 3

[0150] Specific Inhibition of Gene Expression by RNA Interference

[0151] 3.1 Materials and Methods

[0152] 3.1.1 RNA Preparation and RNAi Assay

[0153] Chemical RNA synthesis, annealing, and luciferase-based RNAiassays were performed as described in Examples 1 or 2 or in previouspublications (Tuschl et al., 1999; Zamore et al., 2000). All siRNAduplexes were directed against firefly luciferase, and the luciferasemRNA sequence was derived from pGEM-luc (GenBank acc. X65316) asdescribed (Tuschl et al., 1999). The siRNA duplexes were incubated in D.melanogaster RNAi/translation reaction for 15 min prior to addition ofmRNAs. Translation-based RNAi assays were performed at least intriplicates.

[0154] For mapping of sense target RNA cleavage, a 177-nt transcript wasgenerated, corresponding to the firefly luciferase sequence betweenpositions 113-273 relative to the start codon, followed by the 17-ntcomplement of the SP6 promoter sequence. For mapping of aritisensetarget RNA cleavage, a 166-nt transcript was produced from a template,which was amplified from plasmid sequence by PCR using 5′ primerTAATACGACTCACTATAGAGCCCATATCGTTTCATA (T7 promoter underlined) and 3′primer AGAGGATGGAACCGCTGG. The target sequence corresponds to thecomplement of the firefly luciferase sequence between positions 50-215relative to the start codon. Guanylyl transferase labelling wasperformed as previously described (Zamore et al., 2000). For mapping oftarget RNA cleavage, 100 nM siRNA duplex was incubated with 5 to 10 nMtarget RNA in D. melanogaster embryo lysate under standard conditions(Zamore et al., 2000) for 2 h at 25° C. The reaction was stopped by theaddition of 8 volumes of proteinase K buffer (200 mM Tris-HCl pH 7.5, 25mM EDTA, 300 mM NaCl, 2% w/v sodium dodecyl sulfate). Proteinase K (E.M.Merck, dissolved in water) was added to a final concentration of 0.6mg/ml. The reactions were then incubated for 15 min at 65° C., extractedwith phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with 3volumes of ethanol. Samples were located on 6% sequencing gels. Lengthstandards were generated by partial RNase T1 digestion and partial basehydrolysis of the cap-labelled sense or antisense target RNAs.

[0155] 3.2 Results

[0156] 3.2.1 Variation of the 3′ Overhang in Duplexes of 21-nt siRNAs

[0157] As described above, 2 or 3 unpaired nucleotides at the 3′ end ofsiRNA duplexes were more efficient in target RNA degradation than therespective blunt-ended duplexes. To perform a more comprehensiveanalysis of the function of the terminal nucleotides, we synthesizedfive 21-nt sense siRNAs, each displayed by one nucleotide relative tothe target RNA, and eight 21-nt antisense siRNAs, each displaced by onenucleotide relative to the target (FIG. 11A). By combining sense andantisense siRNAs, eight series of siRNA duplexes with syntheticoverhanging ends were generated covering a range of 7-nt 3′ overhang to4-nt 5′ overhang. The interference of siRNA duplexes was measured usingthe dual luciferase assay system (Tuschl et al., 1999; Zamore et al.,2000). siRNA duplexes were directed against firefly luciferase mRNA, andsea pansy luciferase mRNA was used as internal control. The luminescenceratio of target to control luciferase activity was determined in thepresence of siRNA duplex and was normalized to the ratio observed in theabsence of dsRNA. For comparison, the interference ratios of long dsRNAs(39 to 504 pb) are shown in FIG. 11B. The interference ratios weredetermined at concentrations of 5 nM for long dsRNAs (FIG. 11A) and at100 nM for siRNA duplexes (FIG. 11C-J). The 100 nM concentrations ofsiRNAs was chosen, because complete processing of 5 nM 504 bp dsRNAwould result in 120 nM total siRNA duplexes.

[0158] The ability of 21-nt siRNA duplexes to mediate RNAi is dependenton the number of overhanging nucleotides or base pairs formed. Duplexeswith four to six 3′ overhanging nucleotides were unable to mediate RNAi(FIG. 11C-F), as were duplexes with two or more 5′ overhangingnucleotides (FIG. 11G-J). The duplexes with 2-nt 3′ overhangs were mostefficient in mediating RNA interference, though the efficiency ofsilencing was also sequence-dependent, and up to 12-fold differenceswere observed for different siRNA duplexes with 2-nt 3′ overhangs(compare FIG. 11D-H). Duplexes with blunted ends, 1-nt 5′ overhang or 1-to 3-nt 3′ overhangs were sometimes functional. The small silencingeffect observed for the siRNA duplex with 7-nt 3′ overhang (FIG. 11C)may be due to an antisense effect of the long 3′ overhang rather thandue to RNAi. Comparison of the efficiency of RNAi between long dsRNAs(FIG. 11B) and the most effective 21-nt siRNA duplexes (FIG. 11E, G, H)indicates that a single siRNA duplex at 100 nM concentration can be aseffective as 5 nM 504 bp dsRNA.

[0159] 3.2.2 Length Variation of the Sense siRNA Paired to an Invariant21-nt Antisense siRNA

[0160] In order to investigate the effect of length of siRNA on RNAi, weprepared 3 series of siRNA duplexes, combining three 21-nt antisensestrands with eight, 18- to 25-nt sense strands the 3′ overhang of theantisense siRNA was fixed to 1, 2, or 3 nt in each siRNA duplex series,while the sense siRNA was varied at its 3′ end (FIG. 12A). Independentof the lenght of the sense siRNA, we found that duplexes with 2-nt 3′overhang of antisense siRNA (FIG. 12C) were more active than those with1- or 3-nt 3′ overhang (FIG. 12B, D). In the first series, with 1-nt 3′overhang of antisense siRNA, duplexes with a 21- and 22-nt sense siRNAs,carrying a 1- and 2-nt 3‘overhang of sense’ siRNA, respectively, weremost active. Duplexes with 19- to 25-nt sense siRNAs were also able tomediate RNA, but to a lesser extent. Similarly, in the second series,with 2-nt overhang of antisense siRNA, the 21-nt siRNA duplex with 2-nt3′ overhang was most active, and any other combination with the 18- to25-nt sense siRNAs was active to a significant degree. In the lastseries, with 3-nt antisense siRNA 3′ overhang, only the duplex with a20-nt sense siRNA and the 2-nt sense 3′ overhang was able to reducetarget RNA expression. Together, these results indicate that the lengthof the siRNA as well as the length of the 3′ overhang are important, andthat duplexes of 21-nt siRNAs with 2-nt 3′ overhang are optimal forRNAi.

[0161] 3.2.3 Length Variation of siRNA Duplexes with a Constant 2-nt 3′Overhang

[0162] We then examined the effect of simultaneously changing the lengthof both siRNA strands by maintaining symmetric 2-nt 3′ overhangs (FIG.13A). Two series of siRNA duplexes were prepared including the 21-ntsiRNA duplex of FIG. 11H as reference. The length of the duplexes wasvaried between 20 to 25 bp by extending the base-paired segment at the3′ end of the sense siRNA (FIG. 13B) or at the 3′ end of the antisensesiRNA (FIG. 13C). Duplexes of 20 to 23 bp caused specific repression oftarget luciferase activity, but the 21-nt siRNA duplex was at least8-fold more efficient than any of the other duplexes. 24- and 25-ntsiRNA duplexes did not result in any detectable interference.Sequence-specific effects were minor as variations on both ends of theduplex produced similar effects.

[0163] 3.2.4 2′-Deoxy and 2′-O-Methyl-Modified siRNA Duplexes

[0164] To assess the importance of the siRNA ribose residues for RNAi,duplexes with 21-nt siRNAs and 2-nt 3′ overhangs with 2′-deoxy- or2′-O-methyl-modified strands were examined (FIG. 14). Substitution ofthe 2-nt 3′ overhangs by 2′-deoxy nucleotides had no effect, and eventhe replacement of two additional riboncleotides adjacent to theoverhangs in the paired region, produced significantly active siRNAs.Thus, 8 out of 42 nt of a siRNA duplex were replaced by DNA residueswithout loss of activity. Complete substitution of one or both siRNAstrands by 2′-deoxy residues, however, abolished RNAi, as didsubstitution by 2′-O-methyl residues.

[0165] 3.2.5 Definition of Target RNA Cleavage Sites

[0166] Target RNA cleavage positions were previously determined for22-nt siRNA duplexes and for a 21-rit/22-nt duplex. It was found thatthe position of the target RNA cleavage was located in the centre of theregion covered by the siRNA duplex, 11 or 12 nt downstream of the firstnucleotide that was complementary to the 21- or 22-nt siRNA guidesequence. Five distinct 21-nt siRNA duplexes with 2-nt 3′ overhang (FIG.15A) were incubated with 5′ cap-labelled sense or antisense target RNAin D. melanogaster lysate (Tuschl et al., 1999; Zamore et al., 2000).The 5′ cleavage products were resolved on sequencing gels (FIG. 15B).The amount of sense target RNA cleaved correlates with the efficiency ofsiRNA duplexes determined in the translation-based assay, and siRNAduplexes 1, 2 and 4 (FIG. 15B and 11H, G, E) cleave target RNA fasterthan duplexes 3 and 5 (FIGS. 15B and 11F, D). Notably, the sum ofradioactivity of the 5′ cleavage product and the input target RNA werenot constant over time, and the 5′ cleavage products did not accumulate.Presumably, the cleavage products, once released from thesiRNA-endonuclease complex, are rapidly degraded due to the lack ofeither of the poly(A) tail of the 5′-cap.

[0167] The cleavage sites for both, sense and antisense target RNAs werelocated in the middle of the region spanned by the siRNA duplexes. Thecleavage sites for each target produced by the 5 different duplexesvaried by 1-nt according to the 1-nt displacement of the duplexes alongthe target sequences. The targets were cleaved precisely 11 ntdownstream of the target position complementary to the 3′-mostnucleotide of the sequence-complementary guide siRNA (FIG. 15A, B).

[0168] In order to determine, whether the 5′ or the 3′ end of the guidesiRNA sets the ruler for target RNA cleavage, we devised theexperimental strategy outlined in FIGS. 16A and B. A 21-nt antisensesiRNA, which was kept invariant for this study, was paired with sensesiRNAs that were modified at either of their 5′ or 3′ ends. The positionof sense and antisense target RNA cleavage was determined as describedabove. Changes in the 3′ end of the sense siRNA, monitored for 1-nt 5′overhang to 6-nt 3′ overhang, did neither effect the position of sensenor antisense target RNA cleavage (FIG. 16C). Changes in the 5′ end ofthe sense siRNA did no affect the sense target RNA cleavage (FIG. 16D,top panel), which was expected because the antisense siRNA wasunchanged. However, the antisense target RNA cleavage was affected andstrongly dependent on the 5′ end of the sense siRNA (FIG. 16D, bottompanel). The antisense target was only cleaved, when the sense siRNA was20 or 21 nt in size, and the position of cleavage different by 1-nt,suggesting that the 5′ end of the target-recognizing siRNA sets theruler for target RNA cleavage. The position is located betweennucleotide 10 and 11 when counting in upstream direction from the targetnucleotide paired to the 5′-most nucleotide of the guide siRNA (see alsoFIG. 15A).

[0169] 3.2.6 Sequence Effects and 2′-Deoxy Substitutions in the 3′Overhang

[0170] A 2-nt 3′overhang is preferred for siRNA function. We wanted toknow, if the sequence of the overhanging nucleotides contributes totarget recognition, or if it is only a feature required forreconstitution of the endonuclease complex (RISC or siRNP). Wesynthesized sense and antisense siRNAs with AA, CC, GG, UU, and UG 3′overhangs and included the 2‘-’ deoxy modifications TdG and TT. Thewild-type siRNAs contained AA in the sense 3′ overhang and UG in theantisense 3′ overhang (AA/UG). All siRNA duplexes were functional in theinterference assay and reduced target expression at least 5-fold (FIG.17). The most efficient siRNA duplexes that reduced target expressionmore than 10-foldi were of the sequence type NN/UG, NN/UU, NN/TdG, andNN/TT (N, any nucleotide) siRNA duplexes with an antisense siRNA 3′overhang of AA, CC or GG were less active by a factor 2 to 4 whencompared to the wild-type sequence UG or the mutant UU. This reductionin RNAi efficiency is likely due to the contribution of the penultimate3′ nudleotide to sequence-specific target recognition, as the 3′terminal nucleotide was changed from G to U without effect.

[0171] Changes in the sequence of the 3′ overhang of the sense siRNA didnot reveal any sequence-dependent effects, which was expected, becausethe sense siRNA must not contribute to sense target mRNA recognition.

[0172] 3.2.7 Sequence Specifity of Target Recognition

[0173] In order to examine the sequence-specifity of target recognition,we introduced sequence changes into the paired segments of siRNAduplexes and determined the efficiency of silencing. Sequence changeswere introduced by inverting short segments of 3- or 4-nt length or aspoint mutations (FIG. 18). The sequence changes in one siRNA strand werecompensated in the complementary siRNA strand to avoid pertubing thebase-paired siRNA duplex structure. The sequence of all 2-nt 3′overhangs was TT (T, 2′-deoxythymidine) to reduce costs of synthesis.The TT/TT reference siRNA duplex was comparable in RNAi to the wild-typesiRNA duplex AA/UG (FIG. 17). The ability to mediate reporter mRNAdestruction was quantified using the translation-based luminescenceassay. Duplexes of siRNAs with inverted sequence segments showeddramatically reduced ability for targeting the firefly luciferasereporter (FIG. 18). The sequence changes located between the 3′ end andthe middle of the antisense siRNA completely abolished target RNArecognition, but mutations near the 5′ end of the antisense siRNAexhibit a small degree of silencing. Transversion of the A/U base pairlocated directly opposite of the predicted target RNA cleavage site, orone nucleotide further away from the predicted site, prevented targetRNA cleavage, therefore indicating that single mutation within thecentre of a siRNA duplex discriminate between mismatched targets.

[0174] 3.3 Discussion

[0175] siRNAs are valuable reagents for inactivation of gene expression,not only in insect cells, but also in mammalian cells, with a greatpotential for therapeutic application. We have systematically analysedthe structural determinants of siRNA duplexes required to promoteefficient target RNA degradation in D. melanogaster embryo lysate, thusproviding rules for the design of most potent siRNA duplexes. A perfectsiRNA duplex is able to silence gene expression with an efficiencycomparable to a 500 bp dsRNA, given that comparable quantities of totalRNA are used.

[0176] 3.4 The siRNA User Guide

[0177] Efficiently silencing siRNA duplexes are preferably composed of21-nt antisense siRNAs, and should be selected to form a 19 bp doublehelix with 2-nt 3′ overhanging ends. 2′-deoxy substitutions of the 2-nt3′ overhanging ribonucleotides do not affect RNAi, but help to reducethe costs of RNA synthesis and may enhance RNAse resistance of siRNAduplexes. More extensive 2′-deoxy or 2′-O-methyl modifications, however,reduce the ability of siRNAs to mediate RNAi, probably by interferingwith protein association for siRNAP assembly.

[0178] Target recognition is a highly sequence-specific process,mediated by the siRNA complementary to the target. The 3′-mostnucleotide of the guide siRNA does not contribute to specificity oftarget recognition, while the penultimate nucleotide of the 3′ overhangaffects target RNA cleavage, and a mismatch reduces RNAi 2- to 4-fold.The 5′ end of a guide siRNA also appears more permissive for mismatchedtarget RNA recognition when compared to the 3′ end. Nucleotides in thecentre of the siRNA, located opposite the target RNA cleavage site, areimportant specificity determinants and even single nucleotide changesreduce RNAi to undetectable level. This suggests that siRNA duplexes maybe able to discriminate mutant or polymorphic alleles in gene targetingexperiments, which may become an important feature for futuretherapeutic developments.

[0179] Sense and antisense siRNAs, when associated with the proteincomponents of the endonclease complex or its commitment complex, weresuggested to play distinct roles; the relative orientation of the siRNAduplex in this complex defines which strand can be used for targetrecognition. Synthetic siRNA duplexes have dyad symmetry with respect tothe doublehelical structure, but not with respect to sequence. Theassociation of siRNA duplexes with the RNAi proteins in the D.melanogaster lysate will lead to formation of two asymmetric complexes.In such hypothetical complexes, the chiral environment is distinct forsense and antisense siRNA, hence their function. The predictionobviously does not apply to palindromic siRNA sequences, or to RNAiproteins that could associate as homodimers. To minimize sequenceeffects, which may affect the ratio of sense and antisense-targetingsiRNPs, we suggest to use siRNA sequences with identical 3′ overhangingsequences. We recommend to adjust the sequence of the overhang of thesense siRNA to that of the antisense 3′ overhang, because the sensesiRNA does not have a target in typical knock-down experiments.Asymmetry in reconstitution of sense and anti-sense-cleaving siRNPscould be (partially) responsible for the variation in RNAi efficiencyobserved for various 21-nt siRNA duplexes with 2-nt 3′ overhangs used inthis study (FIG. 14). Alternatively, the nucleotide sequence at thetarget site and/or the accessibility of the target RNA structure may beresponsible for the variation in efficiency for these siRNA duplexes.

REFERENCES

[0180] Bass, B. L. (2000). Double-stranded RNA as a template for genesilencing. Cell 101, 235-238.

[0181] Bosher, J. M., and Labouesse, M. (2000). RNA interference:genetic wand and genetic watchdog. Nat. Cell Biol. 2, E31-36.

[0182] Caplen, N. J., Fleenor, J., Fire, A., and Morgan, R. A. (2000).dsRNA-mediated gene silencing in cultured Drosophila cells: a tissueculture model for the analysis of RNA interference. Gene 252, 95-105.

[0183] Catalanotto, C.; Azzalin, G., Macino, G., and Cogoni, C. (2000).Gene silencing in worms and fungi. Nature 404, 245.

[0184] Chanfreau, G., Buckle, M., and Jacquier, A. (2000). Recognitionof a conserved class of RNA tetraloops, by Saccharomyces cerevisiaeRNase III. Proc. Natl. Acad. Sci. USA 97, 3142-3147.

[0185] Clemens, M. J. (1997). PKR-a protein kinase regulated bydouble-stranded RNA. Int. J. Biochem. Cell Biol. 29, 945-949.

[0186] Cogoni, C., and Macino, G. (1999). Homology-dependent genesilencing in plants and fungi: a number of variations on the same theme.Curr. Opin. Microbiol. 2, 657-662.

[0187] Dalmay, T., Hamilton, A., Rudd, S., Angell, S., and Baulcombe, D.C. (2000). An RNA-dependent RNA polymerase gene in Arabidopsis isrequired for posttranscriptional gene silencing mediated by a transgenebut not by a virus. Cell 101, 543-553.

[0188] Dernburg, A. F., Zalevsky, J., Colaiacovo, M. P., and Villeneuve,A. M. (2000). Transgene-mediated cosuppression in the C. elegans germline. Genes & Dev. 14, 1578-1583.

[0189] Dunn, J. J. (1982). Ribonuclease III. In The enzymres, vol 15,part B, P. D. Boyer, ed. (New York: Academic Press), pp. 485-499.

[0190] Filippov, V., Solovyev, V., Filippova, M., and Gill, S. S.(2000). A novel type of RNase III family proteins in eukaryotes. Gene245, 213-221.

[0191] Fire, A. (1999). RNA-triggered gene silencing. Trends Genet. 15,358-363.

[0192] Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S.E., and Mello, C. C. (1998). Potent and specific genetic interference bydouble-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.

[0193] Grishok, A., Tabara, H., and Mello, C. C. (2000). Geneticrequirements for inheritance of RNAi in C. elegans. Science 287,2494-2497.

[0194] Hamilton, A. J., and Baulcombe, D. C. (1999). A species of smallanti-sense RNA in posttranscriptional gene silencing in plants. Science286, 950-952.

[0195] Hammond, S. M., Bernstein, E., Beach, D., and Hannon, G. J.(2000). An RNA-directed nuclease mediates post-transcriptional genesilencing in Drosophila cells. Nature 404, 293-296.

[0196] Jacobsen, S. E., Running, M. P., and M., M. E. (1999). Disruptionof an RNA helicase/RNase III gene in Arabidopsis causes unregulated celldivision in floral meristems. Development 126, 5231-5243.

[0197] Jensen, S., Gassama, M. P., and Heidmann, T. (1999). Taming oftransposable elements by homology-dependent gene silencing. Nat. Genet.21, 209-212.

[0198] Kehlenbach, R. H., Dickmanns, A. & Gerace, L. (1998).Nucleocytoplasmic shuttling factors including Ran and CRM1 mediatenuclear export of NFAT In vitro. J. Cell Biol. 141, 863-874.

[0199] Kennerdell, J. R., and Carthew, R. W. (1998). Use ofdsRNA-mediated genetic interference to demonstrate that frizzled andfrizzled 2 act in the wingless pathway. Cell 95, 1017-1026.

[0200] Ketting, R. F., Haverkamp, T. H., van Luenen, H. G., andPlasterk, R. H. (1999). Mut-7 of C. elegans, required for transposonsilencing and RNA interference, is a homblog of Werner syndrome helicaseand RNaseD. Cell 99, 133-141.

[0201] Ketting, R. F., and Plasterk, R. H. (2000). A genetic linkbetween co-suppression and RNA interference in C. elegans. Nature 404,296-298.

[0202] Lucy, A. P., Guo, H. S., Li, W. X., and Ding, S. W. (2000).Suppression of post-transcriptional gene silencing by a plant viralprotein localized in the nucleus. EMBO J. 19, 1672-1680.

[0203] Matsuda, S., Ichigotani, Y., Okuda, T., Irimura, T., Nakatsugawa,S., and Hamaguchi, M. (2000). Molecular cloning and characterization ofa novel human gene (HERNA) which encodes a putative RNA-helicase.Biochim. Biophys. Acta 31, 1-2.

[0204] Milligan, J. F., and Uhlenbeck, O. C. (1989). Synthesis of smallRNAs using T7 RNA polymerase. Methods Enzymol. 180, 51-62.

[0205] Mourrain, P., Beclin, C., Elmrayan, T., Feuerbach, F., Godon, C.,Morel, J. B., Jouette, D., Lacombe, A. M., Nikic, S., Picault, N.,Remoue, K., Sanial, M., Vo; T. A., and Vaucheret, H. (2000). ArabidopsisSGS2 and SGS3 genes are required for posttranscriptional gene silencingand natural virus resistance. Cell 101, 533-542.

[0206] Ngo, H., Tschudi, C., Gull, K., and Ullu, E. (1998).Double-stranded RNA induces mRNA degradation in Trypanosoma brucei.Proc. Natl. Acad. Sci. USA 95, 14687-14692.

[0207] Nicholson, A. W. (1999). Function, mechanism and regulation ofbacterial ribonucleases. FEMS Microbiol. Rev. 23, 371-390.

[0208] Oelgeschlager, M., Larrain, J., Geissert, D., and De Robertis, E.M. (2000). The evolutionarily conserved BMP-binding protein Twistedgastrulation promotes BMP signalling. Nature 405, 757-763.

[0209] Pan, T., and Uhlenbeck, O. C. (1992). In vitro selection of RNAsthat undergo autolytic cleavage with Pb²⁺. Biochemistry 31, 3887-3895.

[0210] Pelissier, T., and Wassenegger, M. (2000). A DNA target of 30 bpis sufficient for RNA-directed methylation. RNA 6, 55-65.

[0211] Plasterk, R. H., and Ketting, R. F. (2000). The silence of thegenes. Curr. Opin. Genet. Dev. 10, 562-567.

[0212] Ratcliff, F. G., MacFarlane, S. A., and Baulcombe, D. C. (1999).Gene Silencing without DNA. RNA-mediated cross-protection betweenviruses. Plant Cell 11, 1207-1216.

[0213] Robertson, H. D. (1990). Escherichia coli ribonuclease III.Methods Enzymol. 181, 189-202.

[0214] Robertson, H. D. (1982). Escherichia coli ribonuclease IIIcleavage sites. Cell 30, 669-672.

[0215] Romaniuk, E., McLaughlin, L. W., Neilson, T., and Romaniuk, P. J.(1982). The effect of acceptor oligoribonucleotide sequence on the T4RNA ligase reaction. Eur J Biochem 125, 639-643.

[0216] Sharp, P. A. (1999). RNAi and double-strand RNA. Genes & Dev. 13,139-141.

[0217] Sijen, T., and Kooter, J. M. (2000). Post-transcriptionalgene-silencing: RNAs on the attack or on the defense? Bioessays 22,520-531.

[0218] Smardon, A., Spoerke, J., Stacey, S., Klein, M., Mackin, N., andMaine, E. (2000). EGO-1 is related to RNA-directed RNA polymerase andfunctions in germ-line development and RNA interference in C. elegans.Curr. Biol. 10, 169-178.

[0219] Svoboda, P., Stein, P., Hayashi, H., and Schultz, R. M. (2000).Selective reduction of dormant maternal mRNAs in mouse oocytes by RNAinterference. Development 127, 4147-4156.

[0220] Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok,A., Timmons, L., Fire, A., and Mello, C. C. (1999). The rde-1 gene, RNAinterference, and transposon silencing in C. elegans. Cell 99, 123-132.

[0221] Tuschl, T., Ng, M. M., Pieken, W., Benseleri F.; and Eckstein, F.(1993). Importance of exocyclic base functional groups of central coreguanosines for hammerhead ribozyme activity. Biochemistry 32,11658-11.668.

[0222] Tuschl, T., Sharp, P. A., and Bartel, D. P. (1998). Selection invitro of novel ribozymes from a partially randomized U2 and U6 snRNAlibrary. EMBO J. 17, 2637-2650.

[0223] Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P., and Sharp,P. A. (1999). Targeted mRNA degradation by double-stranded RNA in vitro.Genes & Dev. 13, 3191-3197.

[0224] Ui-Tei, K., Zenno, S., Miyata, Y. & Saigo, K. (2000). Sensitiveassay of RNA interference in Drosophila and Chinese hamster culturedcells using firefly luciferase gene as target. FEBS Letters 479, 79-82.

[0225] Verma, S., and Eckstein, F. (1999). Modified oligonucleotides:Synthesis and strategy for users. Annu. Rev. Biochem. 67, 99-134.

[0226] Voinnet, O., Lederer, C., and Baulcombe, D. C. (2000). A viralmovement protein prevents spread of the gene silencing signal in.Nicotiana benthamiana. Cell 103, 157-167.

[0227] Wassenegger, M. (2000). RNA-directed DNA methylation. Plant Mol.Biol. 43, 203-220.

[0228] Wianny, F., and Zernicka-Goetz, M. (2000). Specific interferencewith gene function by double-stranded RNA in early mouse development.Nat. Cell Biol. 2, 70-75.

[0229] Wu, H., Xu, H., Miraglia, L. J., and Crooke, S. T. (2000). HumanRNase III is a 160 kDa Protein Involved in Preribosomal RNA Processing.J. Biol. Chem. 17, 17.

[0230] Yang, D., Lu, H. and Erickson, J. W. (2000) Evidence thatprocessed small dsRNAs may mediate sequence-specific mRNA degradationduring RNAi in drosophilia embryos. Curr. Biol., 10, 1191-1200.

[0231] Zamore, P. D., Tuschl, T., Sharp, P. A., and Bartel, D. P.(2000). RNAi: Double-stranded RNA directs the ATP-dependent cleavage ofmRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33.

[0232] Zhang, K., and Nicholson, A. W. (1997). Regulation ofribonuclease III processing by double-helical sequence antideterminants.Proc. Natl. Acad. Sci. USA 94, 13437-13441.

1 101 1 38 DNA Artificial sequence pGEM-luc sequence from the Pp-lucplasmid 1 gcgtaatacg actcactata gaacaattgc ttttacag 38 2 35 DNAArtificial Sequence pGEM-luc sequence from the Pp-luc plasmid 2atttaggtga cactataggc ataaagaatt gaaga 35 3 30 DNA Artificial sequencereverse transcription primer for cloning RNAs 3 gactagctgg aattcaaggatgcggttaaa 30 4 30 DNA Artificial sequence PCR primer for cloning RNAs 4cagccaacgg aattcatacg actcactaaa 30 5 36 DNA Artificial sequence PCRprimer that amplifies firefly luciferase sequence in a plasmid 5taatacgact cactatagag cccatatcgt ttcata 36 6 18 DNA Artificial sequencePCR primer that amplifies firefly luciferase sequence in a plasmid 6agaggatgga accgctgg 18 7 177 RNA Artificial sequence luciferase gene 7gaacaauugc uuuuacagau gcacauaucg aggugaacau cacguacgcg gaauacuucg 60aaauguccgu ucgguuggca gaagcuauga aacgauaugg gcugaauaca aaucacagaa 120ucgucguaug cagugaaaac ucucuucaau ucuuuaugcc uauaguguca ccuaaau 177 8 180RNA Artificial sequence luciferase gene 8 ggcauaaaga auugaagagaguuuucacug cauacgacga uucugugauu uguauucagc 60 ccauaucguu ucauagcuucugccaaccga acggacauuu cgaaguauuc cgcguacgug 120 auguucaccu cgauaugugcaucuguaaaa gcaauuguuc uauagugagu cguauuacgc 180 9 39 RNA Artificialsequence luciferase gene 9 gcacauaucg aggugaacau cacguacgcg gaauacuuc 3910 52 RNA Artificial sequence luciferase gene 10 gcacauaucg aggugaacaucacguacgcg gaauacuucg aaauguccgu uc 52 11 111 RNA Artificial sequenceluciferase gene 11 gcacauaucg aggugaacau cacguacgcg gaauacuucgaaauguccgu ucgguuggca 60 gaagcuauga aacgauaugg gcugaauaca aaucacagaaucgucguaug c 111 12 52 RNA Artificial sequence luciferase gene 12gcacauaucg aggugaacau cacguacgcg gaauacuucg aaauguccgu uc 52 13 54 RNAArtificial sequence luciferase gene 13 gaacggacau uucgaaguau uccgcguacgugauguucac cucgauaugu gcac 54 14 21 RNA Artificial sequence luciferasegene 14 cguacgcgga auacuucgau u 21 15 21 RNA Artificial sequenceluciferase gene 15 ucgaaguauu ccgcguacgu u 21 16 21 DNA Artificialsequence luciferase gene 16 cguacgcgga auacuucgat t 21 17 21 DNAArtificial sequence luciferase gene 17 ucgaaguauu ccgcguacgt t 21 18 21DNA Artificial sequence luciferase gene 18 cuuacgcuga guacuucgat t 21 1921 DNA Artificial sequence luciferase gene 19 ucgaaguacu cagcguaagt t 2120 21 DNA Artificial sequence luciferase gene 20 agcuucauaa ggcgcaugct t21 21 21 DNA Artificial sequence luciferase gene 21 gcaugcgccuuaugaagcut t 21 22 21 DNA Artificial sequence luciferase gene 22aaacaugcag aaaaugcugt t 21 23 21 DNA Artificial sequence luciferase gene23 cagcauuuuc ugcauguuut t 21 24 21 RNA Artificial sequence luciferasegene 24 aucacguacg cggaauacuu c 21 25 21 RNA Artificial sequenceluciferase gene 25 guauuccgcg uacgugaugu u 21 26 21 RNA Artificialsequence luciferase gene 26 ucacguacgc ggaauacuuc g 21 27 21 RNAArtificial sequence luciferase gene 27 cacguacgcg gaauacuucg a 21 28 21RNA Artificial sequence luciferase gene 28 acguacgcgg aauacuucga a 21 2921 RNA Artificial sequence luciferase gene 29 cguacgcgga auacuucgaa a 2130 21 RNA Artificial sequence luciferase gene 30 aguauuccgc guacgugaug u21 31 21 RNA Artificial sequence luciferase gene 31 aaguauuccgcguacgugau g 21 32 21 RNA Artificial sequence luciferase gene 32gaaguauucc gcguacguga u 21 33 21 RNA Artificial sequence luciferase gene33 cgaaguauuc cgcguacgug a 21 34 21 RNA Artificial sequence luciferasegene 34 ucgaaguauu ccgcguacgu g 21 35 21 RNA Artificial sequenceluciferase gene 35 uucgaaguau uccgcguacg u 21 36 21 RNA Artificialsequence luciferase gene 36 uuucgaagua uuccgcguac g 21 37 18 RNAArtificial sequence luciferase gene 37 cguacgcgga auacuucg 18 38 21 RNAArtificial sequence luciferase gene 38 uucgaaguau uccgcguacg u 21 39 19RNA Artificial sequence luciferase gene 39 cguacgcgga auacuucga 19 40 20RNA Artificial sequence luciferase gene 40 cguacgcgga auacuucgaa 20 4121 RNA Artificial sequence luciferase gene 41 cguacgcgga auacuucgaa a 2142 22 RNA Artificial sequence luciferase gene 42 cguacgcgga auacuucgaaau 22 43 23 RNA Artificial sequence luciferase gene 43 cguacgcggaauacuucgaa aug 23 44 24 RNA Artificial sequence luciferase gene 44cguacgcgga auacuucgaa augu 24 45 25 RNA Artificial sequence luciferasegene 45 cguacgcgga auacuucgaa auguc 25 46 21 RNA Artificial sequenceluciferase gene 46 ucgaaguauu ccgcguacgu g 21 47 21 RNA Artificialsequence luciferase gene 47 cgaaguauuc cgcguacgug a 21 48 20 RNAArtificial sequence luciferase gene 48 cguacgcgga auacuucgaa 20 49 20RNA Artificial sequence luciferase gene 49 cgaaguauuc cgcguacgug 20 5021 RNA Artificial sequence luciferase gene 50 cguacgcgga auacuucgaa a 2151 21 RNA Artificial sequence luciferase gene 51 ucgaaguauu ccgcguacgu g21 52 22 RNA Artificial sequence luciferase gene 52 cguacgcggaauacuucgaa au 22 53 22 RNA Artificial sequence luciferase gene 53uucgaaguau uccgcguacg ug 22 54 23 RNA Artificial sequence luciferasegene 54 cguacgcgga auacuucgaa aug 23 55 23 RNA Artificial sequenceluciferase gene 55 uuucgaagua uuccgcguac gug 23 56 24 RNA Artificialsequence luciferase gene 56 cguacgcgga auacuucgaa augu 24 57 24 RNAArtificial sequence luciferase gene 57 auuucgaagu auuccgcgua cgug 24 5825 RNA Artificial sequence luciferase gene 58 cguacgcgga auacuucgaaauguc 25 59 25 RNA Artificial sequence luciferase gene 59 cauuucgaaguauuccgcgu acgug 25 60 19 RNA Artificial sequence luciferase gene 60guacgcggaa uacuucgaa 19 61 20 RNA Artificial sequence luciferase gene 61ucgaaguauu ccgcguacgu 20 62 22 RNA Artificial sequence luciferase gene62 acguacgcgg aauacuucga aa 22 63 22 RNA Artificial sequence luciferasegene 63 ucgaaguauu ccgcguacgu ga 22 64 23 RNA Artificial sequenceluciferase gene 64 cacguacgcg gaauacuucg aaa 23 65 23 RNA Artificialsequence luciferase gene 65 ucgaaguauu ccgcguacgu gau 23 66 21 RNAArtificial sequence luciferase gene 66 acguacgcgg aauacuucga a 21 67 21RNA Artificial sequence luciferase gene 67 cgaaguauuc cgcguacgug a 21 6821 RNA Artificial sequence luciferase gene 68 cacguacgcg gaauacuucg a 2169 21 RNA Artificial sequence luciferase gene 69 gaaguauucc gcguacguga u21 70 21 RNA Artificial sequence luciferase gene 70 ucacguacgcggaauacuuc g 21 71 21 RNA Artificial sequence luciferase gene 71aaguauuccg cguacgugau g 21 72 21 RNA Artificial sequence luciferase gene72 aucacguacg cggaauacuu c 21 73 21 RNA Artificial sequence luciferasegene 73 aguauuccgc guacgugaug u 21 74 18 RNA Artificial sequenceluciferase gene 74 acgcggaaua cuucgaaa 18 75 21 RNA Artificial sequenceluciferase gene 75 ucgaaguauu ccgcguacgu g 21 76 19 RNA Artificialsequence luciferase gene 76 uacgcggaau acuucgaaa 19 77 20 RNA Artificialsequence luciferase gene 77 guacgcggaa uacuucgaaa 20 78 21 RNAArtificial sequence luciferase gene 78 cguacgcgga auacuucgaa a 21 79 22RNA Artificial sequence luciferase gene 79 acguacgcgg aauacuucga aa 2280 23 RNA Artificial sequence luciferase gene 80 cacguacgcg gaauacuucgaaa 23 81 21 DNA Artificial sequence luciferase gene 81 cguacgcggaauacuucgat t 21 82 21 DNA Artificial sequence luciferase gene 82ucgaaguauu ccgcguacgt t 21 83 21 DNA Artificial sequence luciferase gene83 augccgcgga auacuucgat t 21 84 21 DNA Artificial sequence luciferasegene 84 ucgaaguauu ccgcggcaut t 21 85 21 DNA Artificial sequenceluciferase gene 85 cguagcgcga auacuucgat t 21 86 21 DNA Artificialsequence luciferase gene 86 ucgaaguauu cgcgcuacgt t 21 87 21 DNAArtificial sequence luciferase gene 87 cguacgcgag uaacuucgat t 21 88 21DNA Artificial sequence luciferase gene 88 ucgaaguuac ucgcguacgt t 21 8921 DNA Artificial sequence luciferase gene 89 cguacgcgga auuucacgat t 2190 21 DNA Artificial sequence luciferase gene 90 ucgugaaauu ccgcguacgt t21 91 21 DNA Artificial sequence luciferase gene 91 cguacgcggaauacuuagct t 21 92 21 DNA Artificial sequence luciferase gene 92gcuaaguauu ccgcguacgt t 21 93 21 DNA Artificial sequence luciferase gene93 cguacgcggu auacuucgat t 21 94 21 DNA Artificial sequence luciferasegene 94 ucgaaguaua ccgcguacgt t 21 95 21 DNA Artificial sequenceluciferase gene 95 cguacgcgga uuacuucgat t 21 96 21 DNA Artificialsequence luciferase gene 96 ucgaaguaau ccgcguacgt t 21 97 39 RNAArtificial sequence luciferase gene 97 gaaguauucc gcguacguga uguucaccucgauaugugc 39 98 52 RNA Artificial sequence luciferase gene 98 gaacggacauuucgaaguau uccgcguacg ugauguucac cucgauaugu gc 52 99 111 RNA Artificialsequence luciferase gene 99 gcauacgacg auucugugau uuguauucag cccauaucguuucauagcuu cugccaaccg 60 aacggacauu ucgaaguauu ccgcguacgu gauguucaccucgauaugug c 111 100 18 DNA Artificial sequence adapter nucleotide forcloning RNAs 100 uuuaaccgca tccttctc 18 101 20 DNA Artificial sequenceadapter nucleotide for cloning RNAs 101 tactaatacg actcactaaa 20

1. Isolated double-stranded RNA molecule, wherein each RNA strand has alength from 19-25 nucleotides, wherein said RNA molecule is capable oftarget-specific nucleic acid modifications.
 2. The RNA molecule of claim1 wherein at least one strand has a 3′-overhang from 1-5 nucleotides. 3.The RNA molecule of claim 1 capable of target-specific RNA interferenceand/or DNA methylation.
 4. The RNA molecule of claim 1, wherein eachstrand has a length from 19-23, particularly from 20-22 nucleotides. 5.The RNA molecule of claim 2, wherein the 3′-overhang is from 1-3nucleotides.
 6. The RNA molecule of claim 2, wherein the 3′-overhang isstabilized against degradation.
 7. The RNA molecule of claim 1, whichcontains at least one modified nucleotide analogue.
 8. The RNA moleculeof claim 7, wherein the modified nucleotide analogue is selected fromsugar- or backbone modified ribonucleotides.
 9. The RNA moleculeaccording to claim 7, wherein the nucleotide analogue is asugar-modified ribonucleotide, wherein the 2′-OH group is replaced by agroup selected from H, OR, R, halo, SH, SR¹, NH₂, NHR, NR₂ or CN,wherein R is C₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.10. The RNA molecule of claim 7, wherein the nucleotide analogue is abackbone-modified ribonucleotide containing a phosphothioate group. 11.The RNA molecule of claim 1, which has a sequence having an identity ofat least 50 percent to a predetermined mRNA target molecule.
 12. The RNAmolecule of claim 11, wherein the identity is at least 70 percent.
 13. Amethod of preparing a double-stranded RNA molecule of claim 1 comprisingthe steps: (a) synthesizing two RNA strands each having a length from19-25 nucleotides, wherein said RNA strands are capable of forming adouble-stranded RNA molecule, (b) combining the synthesized RNA strandsunder conditions, wherein a double-stranded RNA molecule is formed,which is capable of target-specific nucleic acid modifications.
 14. Themethod of claim 13, wherein the RNA strands are chemically synthesized.15. The method of claim 13, wherein the RNA strands are enzymaticallysynthesized.
 16. A method of mediating target-specific nucleic acidmodifications in a cell or an organism comprising the steps: (a)contacting said cell or organism with the double-stranded RNA moleculeof claim 1 under conditions wherein target-specific nucleic acidmodifications can occur, and (b) mediating a target-specific nucleicacid modification effected by the double-stranded RNA towards a targetnucleic acid having a sequence portion substantially corresponding tothe double-stranded RNA.
 17. The method of claim 16, wherein the nucleicacid modification is RNA interference and/or DNA methylation.
 18. Themethod of claim 16, wherein said contacting comprises introducing saiddouble-stranded RNA molecule into a target cell in which thetarget-specific nucleic acid modification can occur.
 19. The method ofclaim 18 wherein the introducing comprises a carrier-mediated deliveryor injection.
 20. Use of the method of claim 16 or determining thefunction of a gene in a cell or an organism.
 21. Use of the method ofclaim 16 for modulating the function of a gene in a cell or an organism.22. The use of claim 20, wherein the gene is associated with apathological condition.
 23. The use of claim 22, wherein the gene is apathogen-associated gene.
 24. The use of claim 23, wherein the gene is aviral gene.
 25. The use of claim 22, wherein the gene is atumor-associated gene.
 26. The use of claim 22, wherein the gene is anautoimmune disease-associated gene.
 27. Pharmaceutical compositioncontaining as an active agent at least one double-stranded RNA moleculeof claim 1 and a pharmaceutical carrier.
 28. The composition of claim 27for diagnostic applications.
 29. The composition of claim 27 fortherapeutic applications.